WO2004042056A1 - Polypeptide physiologiquement actif et son anticorps ainsi que son utilisation - Google Patents

Polypeptide physiologiquement actif et son anticorps ainsi que son utilisation Download PDF

Info

Publication number
WO2004042056A1
WO2004042056A1 PCT/JP2003/014161 JP0314161W WO2004042056A1 WO 2004042056 A1 WO2004042056 A1 WO 2004042056A1 JP 0314161 W JP0314161 W JP 0314161W WO 2004042056 A1 WO2004042056 A1 WO 2004042056A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
cells
antibody
amino acid
dna
Prior art date
Application number
PCT/JP2003/014161
Other languages
English (en)
Japanese (ja)
Inventor
Makoto Takeuchi
Takanori Okura
Tomoki Tatefuji
Tetsuya Mori
Tsunetaka Ohta
Masashi Kurimoto
Original Assignee
Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo filed Critical Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo
Priority to JP2004549627A priority Critical patent/JPWO2004042056A1/ja
Priority to AU2003277583A priority patent/AU2003277583A1/en
Publication of WO2004042056A1 publication Critical patent/WO2004042056A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • 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
    • 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
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/86—Products or compounds obtained by genetic engineering
    • 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 physiologically active polypeptide, its antibody, and its use.
  • Multicellular organisms such as humans, have the ability to heal wounds and repair body tissues when body tissues are injured or skin cells become inflamed such as by burns. . If such capabilities could be enhanced by some means, it would be possible to repair and treat wounds early, and that means could be useful in a wide range of fields including medicine.
  • MMP matrixmeta II oprotease
  • Gelatinase A (MM P-2) has the action of degrading gelatin and type IV collagen, type V collagen and elastin, which are the main components of the basement membrane between the epithelial cell layer and connective tissue. Gelatinase A is continuously expressed in small amounts in fibroblasts and monocytic cells, and has a physiological effect of suppressing epidermal cell elongation and angiogenesis in cancer tissues. Factors that can effectively enhance the expression of gelatinases such as gelatinase A can be expected to be useful in wound healing and tissue reconstruction, but such factors have not been known in the past.
  • bone marrow transplants which transplant healthy bone marrow cells into patients and produce new and fresh blood, have been frequently performed in order to treat blood diseases such as leukemia and aplastic anemia.
  • blood diseases such as leukemia and aplastic anemia.
  • bone marrow transplantation requires that the types of histocompatibility antigens in patients and bone marrow donors match, and in Europe and the United States, the names of healthy individuals who are willing to provide bone marrow and their The organization of the bone marrow bank, in which the types of bone marrow are registered in advance, began early on, and in Japan, the Bone Marrow Bank Liaison Council was established in 1990, establishing a nationwide network. Making has begun.
  • bone marrow transplantation imposes a considerable burden on the bone marrow donors, both mentally and physically, so even healthy individuals who have previously expressed their intent to donate bone marrow actually request bone marrow donation. In many cases, they are hesitant to refuse or refuse, and as a result, it is said that in medical practice, the necessary bone marrow cannot be obtained when needed.
  • an object of the present invention is to provide a means for effectively enhancing or regulating the expression of gelatinase in mammalian skin cells.
  • the inventors of the present invention focused on DNA derived from mammalian skin cells or placental cells and conducted a research on a physiologically active polypeptide encoded by the DNA, which is shown by any one of SEQ ID NOs: 1 to 3 in the sequence listing.
  • a polypeptide having a partial amino acid sequence is It has been found that when applied to mammalian skin cells in vitro, the expression of gelatinase is remarkably enhanced, and when applied to wounds on living skin, the expression of gelatinase in cells is enhanced, It has been found to promote healing. On the other hand, it has been found that when the polypeptide acts on hematopoietic cells derived from mammals in vitro, the proliferation is significantly promoted. Furthermore, the present inventors have elucidated DNA encoding such a polypeptide, established a method for producing the polypeptide, and established an antibody against the polypeptide, and have completed the present invention.
  • the present invention relates to a polypeptide having a partial amino acid sequence represented by any one of SEQ ID NOs: 1 to 3 in the sequence listing, or a partial amino acid sequence represented by any of SEQ ID NOs: 1 to 3 in the sequence listing.
  • the object of the present invention is to provide a polypeptide having an amino acid sequence in which one or more amino acids are deleted, substituted, Z or added within a range not to substantially lose the intended biological action described above. Is to solve.
  • the present invention solves the above-mentioned problem by providing a DNA encoding such a polypeptide.
  • the present invention provides a method for producing a polypeptide, comprising a step of culturing a cell or a microorganism capable of producing such a polypeptide, and a step of collecting the produced polypeptide from a culture. This solves the above problem.
  • the present invention solves the above-mentioned problem by providing an antibody against such a polypeptide.
  • the present invention provides a method for producing a DNA sequence comprising 10 or more consecutive amino acid sequences represented by any one of SEQ ID NOS: 4 to 10 in the sequence listing.
  • the object is achieved by providing a peptide fragment consisting of amino acid residues.
  • the present invention is based on the discovery of polypeptides that enhance gelatinase expression in mammalian skin cells and / or promote hematopoietic cell proliferation.
  • the polypeptide of the present invention exerts remarkable gelatinase expression enhancing ability in skin cells, and thus is useful in early repair / treatment of wounds in skin and regenerative medicine.
  • the polypeptide of the present invention effectively promotes the proliferation of hematopoietic cells in mammals, including humans. It is useful as an agent for promoting hematopoietic cell proliferation during transplantation and during expansion of hematopoietic cells in vitro.
  • the antibody of the present invention is useful not only for purification and detection of the above-mentioned polypeptide, but also for regulation and suppression of the biological action of the polypeptide of the present invention.
  • FIG. 5 shows the structure of the expression vector pcD—hAgK114aFL.
  • FIG. 2 is a view showing the structure of the expression vector pVL1303-hAgK114aFL.
  • FIG. 3 is a diagram showing the structure of the expression vector pcD—hAgK114bFL.
  • FIG. 4 is a diagram showing the structure of the expression vector pRER—hamAgKI14d2FL.
  • FIG. 5 shows the structure of the expression vector pRER-smAgKI14FL.
  • FIG. 6 is a diagram showing the structure of the expression vector pRER-mAgKI14b.
  • hAgK114- 1 ac DNA D encoding a polypeptide having the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing
  • hAgK114- 1bc DNA coding for a polypeptide having an amino acid sequence represented by SEQ ID NO: 5 in the sequence listing D
  • FLAG DNA encoding FLAG peptide
  • PCMV Site Megalovirus Promoter
  • hamAgK114-1d2DNA DNA encoding the polypeptide having the anoic acid sequence represented by SEQ ID NO: 7 in the sequence listing
  • mAg K114--1 DNA DNA encoding a polypeptide having the amino acid sequence represented by SEQ ID NO: 9 in the sequence listing mAg K1141-b DNA: SEQ ID NO: 1 in the sequence listing 0 DNA encoding a polypeptide having the amino acid sequence represented by
  • Ne0 neomycin resistance gene
  • E F-1 promoter elongation factor 1 alpha promoter dhfr: dehydrofolate reductase gene
  • the present invention relates to a polypeptide having a partial amino acid sequence represented by any one of SEQ ID NOs: 1 to 3 in the sequence listing, or a partial amino acid sequence represented by any of SEQ ID NOs: 1 to 3 in the sequence listing,
  • the present invention relates to a polypeptide having an amino acid sequence in which one or more amino acids have been deleted, substituted and / or added to the extent that the action is not substantially lost.
  • the polypeptide according to the present invention has a partial amino acid sequence as described above and exerts an action of enhancing the expression of gelatinase in skin cells or an action of promoting proliferation of hematopoietic cells in a living body. , Its purity, origin, and preparation method are not limited.
  • skin cells refer to epithelial cells, fibroblasts, mesenchymal cells and other skin cells
  • hematopoietic cells refer to cells containing hematopoietic stem cells and hematopoietic progenitor cells.
  • More specific polypeptides include, for example, those having an amino acid sequence shown in any one of SEQ ID NOs: 4 to 10 in the sequence listing. From gelatinase The present enhancing action or the promoting action of hematopoietic cell proliferation is remarkable, which is extremely useful in the present invention.
  • amino acid sequences represented by SEQ ID NOs: 2, 3 and 4 in the sequence listing are common to the amino acid sequences represented by SEQ ID NOs: 4 and 5, SEQ ID NOs: 6 and 7, and SEQ ID NOs: 8 to 10 in the sequence listing, respectively. Since the polypeptides having the amino acid sequences of SEQ ID NOs: 4 and 5, SEQ ID NOs: 6 and 7, and SEQ ID NOs: 8 to 10 in the sequence listing have the desired biological actions, Sequence numbers 1 to 3 in the sequence listing, which are common partial amino acid sequences, are considered to be deeply involved in the expression of the desired biological activity.
  • polypeptides having the amino acid sequence represented by any one of SEQ ID NOs: 4 to 10 in the sequence listing are merely examples, and the polypeptide referred to in the present invention should not be limited to these.
  • mutants include, for example, deletion of 1 to 10 alanine or a part or all of such alanine within a range not substantially losing biological action.
  • an amino acid sequence substituted with another amino acid such as glycine, and further, 1 to 60 amino acids at the N-terminus, C-terminus or inside the amino acid sequence represented by any one of SEQ ID NOs: 1 to 3 in the sequence listing Having an amino acid sequence obtained by adding an amino acid of It can be obtained by using protein engineering techniques such as specific mutation and random mutation.
  • the presence or absence of the effect of enhancing the expression of gelatinase in mammalian skin cells can be determined, for example, by determining fibroblasts (NHDF) derived from newborn humans, fibroblasts derived from hamsters, or mesenchymal cells derived from mice using the target polypeptide. After culturing in the presence or absence, the expression level of gelatinase in the supernatant of each culture can be determined by enzyme activity measurement, immunological measurement, or the like.
  • NHDF fibroblasts
  • the presence or absence of the effect of promoting the growth of mammalian hematopoietic cells can be determined by, for example, comparing the number of hematopoietic cells in each culture after culturing bone marrow-derived hematopoietic cells in the presence or absence of the target polypeptide. It can be determined by a method such as
  • the polypeptide of the present invention has a partial amino acid sequence represented by any one of SEQ ID NOs: 1 to 3 in the sequence listing, or has a partial amino acid sequence represented by any of SEQ ID NOs: 1 to 3 in the sequence listing,
  • One or more amino acids have a deleted, substituted and / or added amino acid sequence to the extent that action is not substantially lost, and enhance gelatinase expression in mammalian skin cells
  • any polypeptide that promotes the growth of hematopoietic cells including polypeptides created by recombinant DNA technology, polypeptides derived from natural sources, or chemical
  • the polypeptide may be any of those synthesized, for example, and dextran having an average molecular weight of 50,000 to 10,000, for example. Pullulan, artificially chemically modified such as by binding a water-soluble natural polymer or synthetic polymers such as polyethylene glycol (PEG) May be used.
  • the polypeptide having the partial amino acid sequence represented by SEQ ID NO: 1 in the sequence listing is originally a human-derived polypeptide; Are polypeptides derived from hamster and mouse, respectively.
  • Polypeptides having these partial amino acid sequences can be prepared by using recombinant DNA technology to produce transformed cells or transformed microorganisms capable of producing such polypeptides by recombinant DNA technology and culturing them. Then, the polypeptide can be produced in and out of the cells or inside the cells to produce the polypeptide.
  • the DNA in the present invention means DNA coding for the polypeptide of the present invention.
  • any one of SEQ ID NOs: 11 to 17 in a sequence listing including a sequence encoding a polypeptide having an amino acid sequence represented by any of SEQ ID NOs: 4 to 10 in the sequence listing Or one or more bases of the base sequence represented by any of SEQ ID NOs: 11 to 17 in the sequence listing as long as the amino acid sequence to be encoded is not changed.
  • DNA having a nucleotide sequence complementary thereto or a nucleotide sequence complementary thereto is also included.
  • the DNA of the present invention does not matter whether it is naturally derived or artificially synthesized.
  • Natural sources of the DNA of the present invention include, for example, human placental cells, hamster skin cells, and mouse skin cells, from which genes containing the DNA of the present invention can be obtained. . That is, for example, After crushing the placental cells, the whole RNA is prepared by fractionation using sucrose density gradient ultracentrifugation, etc., and this total RNA is treated with oligo (dT) cellulose, oligo (dT) latex, etc. To isolate mRNA. This mRNA is reacted with reverse transcriptase and DNA polymerase on type I to form a double-stranded cDNA, thereby preparing a cDNA library.
  • a transformant is cultured in a nutrient medium, and a transformant containing DNA encoding the polypeptide of the present invention is collected by applying the colony hybridization method to the culture.
  • the DNA of the present invention can be obtained by treating the thus obtained transformant by an ordinary method.
  • cDNA libraries derived from various cells are commercially available, and such commercially available libraries can also be used.
  • the DNA of the present invention for example, chemically synthesizing based on the base sequence represented by any one of SEQ ID NOS: 11 to 17 in the sequence listing or SEQ ID NO: 4 in the sequence listing DNA encoding the amino acid sequence represented by any one of (1) to (10) is inserted into an appropriate vector capable of autonomous replication to obtain a recombinant vector, which is then appropriately introduced into a host, and the resulting transformant is cultured.
  • the cells may be separated from the culture, and a vector containing the DNA may be collected from the cells.
  • Such a DNA is usually introduced into a host in the form of an expression vector comprising the DNA.
  • Expression vectors usually comprise DNA and autonomously replicable vectors, and if DNA is available, are usually relatively easy to prepare using conventional recombinant DNA techniques.
  • Such vectors include, for example, p K ⁇ 2 2 3-3, p GEX—2 pcs, p RL-input, p BT rp 2, p UB l 10, YE pl 3, T i plasmid, Plasmid vectors such as R i plasmid, pBI122, pCDM8, and pRc / CMV, among which the DNA of the present invention is expressed in microorganisms such as Escherichia coli, Bacillus subtilis, and yeast PKK2 2 3-3, pGEX-2T, pRL-138, pBTrp2, pUB110, and YEp ⁇ 3 are also expressed in cells derived from animals and plants.
  • a method generally used in the art is employed. Specifically, first, the DNA-containing gene of the present invention and the vector are cut with a restriction enzyme and / or ultrasonic waves, and then the generated DNA fragment and the vector fragment are ligated. Restriction enzymes that specifically act on nucleotides in the cleavage of genes and vectors, especially type II restriction enzymes, specifically Sau3AI, EcoRI, EcoRV, HindIII, BamHI, Sam3AI The use of all, Xbal, SacI, PstI, etc., makes it easier to ligate the DNA fragment to the vector fragment.
  • the DNA fragment and the vector fragment may be annealed, and then a DNA ligase may be allowed to act in vivo or in vitro.
  • the thus obtained expression vector for use in the present invention can be replicated indefinitely by appropriately introducing it into a host to obtain a transformant and culturing the transformant.
  • Examples of the host cell into which the DNA of the present invention as described above is introduced include Escherichia coli, Bacillus subtilis, yeast, and the like commonly used in the art for producing transformants. Appropriate microorganisms such as molds and cells of invertebrates such as insects, plants and vertebrates can also be used.
  • the host when the host is Escherichia coli or Bacillus subtilis, the host may be cultured in the presence of an expression vector and calcium ions, or by using the combination cell method or the protoplast method. Good.
  • the calcium phosphate method, the electroporation method, the virus infection method, and, if necessary, the DEAE-dextran method, the lipofection method, the microinjection method, etc. May be applied as appropriate.
  • a test may be performed using the presence or absence of the introduced DNA or the ability to produce the polypeptide of the present invention as an index.
  • vectors and transformants described above refer to J. Sambrook et al., Molecular Cloning, 'Laboratory' Manual, 3rd Edition (Cold Spring Harbor, 2001). And various materials and methods commonly used are described in detail.
  • the culture medium used for culturing the transformant usually contains a carbon source, a nitrogen source, minerals, and, if necessary, micronutrients such as amino acids and vitamins, depending on the type of host cell and vector.
  • a supplemented ordinary medium can be used.
  • Individual carbon sources include, for example, starch, starch hydrolyzate, glucose, fructose, sucrose, trehalose, and the like.
  • Nitrogen sources include, for example, ammonia to ammonia, urea, and nitrate.
  • nitrogen-containing inorganic or organic substances such as oil, yeast, yeast extract, defatted soybean, corn steep liquor, and meat dex.
  • pH 2 When the culture is maintained for about 1 to 6 days while maintaining the culture at about 10 to 10, a culture containing the polypeptide of the present invention can be obtained.
  • cells capable of producing the polypeptide of the present invention may be cultured, and the desired polypeptide may be collected from the culture.
  • Individual cells include human-derived cells, for example, HeLa (AT CCCCL-2) cells, a cell line derived from cervical cancer, and a cell line derived from prostate cancer.
  • PC-3 ATCCCRL-1 4 3 5
  • HuCCTI JCRB0425
  • lung cancer-derived cell lines CaIu-3 ATCCHTB
  • KB ATCCCCL-17
  • ovarian epithelial cells CH 0- K1 ATCCCCL-61
  • kidney-derived fibroblasts BHK-21 ATCCCCL-10) cells
  • mouse cells such as NIH-3T3 (ATCCCRL- 1658) Cells and the like.
  • the above cells can be amplified by a solid phase culture method or a liquid culture method generally used in the art for culturing animal cells.
  • amplification is performed in the body of a warm-blooded animal other than a liver by the in vivo cell proliferation method disclosed in Japanese Patent Application Laid-Open No. 54-98307 by the same applicant as the present applicant. be able to.
  • the warm blood animal used may be any one capable of proliferating mammalian cells.
  • Birds such as birds, birds, dogs, cats, monkeys, goats, In the evening, mammals such as porcupines, porcupines, magpies, guinea pigs, rats, hamsters, normal mice, and wild mice can be used.
  • the expanded mammalian cells can be collected, for example, by collecting cells that have grown by floating in the ascites in the abdominal cavity, or by removing and dispersing a subcutaneously grown tumor, and collecting as necessary.
  • the polypeptide of the present invention may be produced by culturing in the presence of an enhancer or the like.
  • polypeptide of the present invention can also be prepared by chemically synthesizing it according to the amino acid sequence shown in any of SEQ ID NOS: 1 to 3 or SEQ ID NOs: 4 to 10 in the sequence listing.
  • a peptide synthesis method a total synthesis method using an automatic peptide synthesizer generally used in the art, or a method in which a peptide fragment is divided into several blocks in advance and enzymatically condensed. Any of the methods for obtaining the desired polypeptide can be advantageously carried out as needed.
  • the crude preparation of the polypeptide of the present invention which is obtained either by preparation by recombinant DNA technology, preparation from natural sources, or preparation by peptide synthesis method, is a gelatinase expression enhancer or hematopoietic cell of mammalian skin cells. Although it can be used as it is as a growth promoter, it is usually used after purification if necessary before use.
  • a gelatinase expression enhancer or hematopoietic cell of mammalian skin cells Although it can be used as it is as a growth promoter, it is usually used after purification if necessary before use.
  • For purification of the polypeptide of the present invention for example, salting-out, dialysis, fractional precipitation, gel filtration chromatography, gel exchange chromatography, For the purification of bioactive polypeptides such as hydrophobic chromatography, affinity chromatography, chromatographic forcing, gel 3? Ice, and ⁇ 3 ⁇ 4Jr?
  • the purified polypeptide may be concentrated and freeze-dried to a liquid or solid state.
  • an antibody against the polypeptide of the present invention can be prepared using the polypeptide of the present invention.
  • the antibody of the present invention encompasses all immunoglobulins against the polypeptide of the present invention, and is not limited to a specific source, class, or form (polyclonal or monoclonal).
  • the antibody of the present invention can be obtained from a warm-blooded animal immunized with a polypeptide containing a part or all of the polypeptide sequence or a cultured cell thereof.
  • the term "can be obtained from a warm-blooded animal or a cultured cell thereof” means that the antibody of the present invention is obtained from a warm-blooded animal itself immunized with a desired polypeptide, and a preparation method. Regardless, it means that immunoglobulins having the same properties as immunoreactivity (described later) with antibodies obtained from the warm-blooded animal are included. Furthermore, the antibody of the present invention can be obtained by white matter engineering, for example, using a humanized antibody, a chimeric antibody, or a human mouse obtained by replacing the immunoglobulin gene with a human derived mouse. Antibodies such as antibodies are also included.
  • immune reaction means a binding reaction between an antibody and a substance that can be recognized by the antibody, that is, a reaction usually called an antigen-antibody reaction. Immunoreactivity refers to the intensity of the immune reaction. Means
  • the immunization may be performed by a conventional method.
  • the polypeptide of the present invention or a fragment thereof may be used alone as an antigen or injected into a vein, intradermally, subcutaneously, or intraperitoneally of a warm-blooded animal together with an appropriate adjuvant. Inoculate and rear for a certain period of time.
  • Temperature for immunization The blood animal is not particularly limited, and can be used for preparing the antibody of the present invention irrespective of the type of warm-blooded animal, male or female, etc., as long as the desired antibody can be produced.
  • rodents such as mice, rats, hamsters, egrets, and guinea pigs
  • mammals including artiodactyls such as goats and sheep
  • birds including pheasants such as chickens and quails
  • the most suitable one may be selected in consideration of the origin of the antigen to be used and the form and use of the antibody to be prepared.
  • the total inoculum of antigen is usually about 5 to 500 g / animal, and divided into 2 to 20 doses at intervals of about 1 to 4 weeks.
  • Immunize generally, the first vaccination is called “primary immunization”, the second vaccination is called “boost”, and the last vaccination is called “final immunization”).
  • the antibody titer in the immunized animal is increased by a conventional method such as an enzyme-antibody method using the same antigen as that used for the immunization. Check.
  • the immunized animal In order to obtain a polyclonal antibody (antiserum), which is one form of the antibody of the present invention, the immunized animal, usually about 1 to 4 weeks after the immunization as described above, is used for the animal species. Serum (antiserum) may be collected from an appropriate site selected accordingly. If the antiserum thus obtained is further subjected to a conventional method for purifying immunoglobulin, if necessary, it can be separated into desired classes such as lgG, lgA, and IgM. You can also get antibodies.
  • the antibodies of the present invention can also be obtained from isolated cells that can produce such antibodies.
  • isolated cell as used herein means a cell in a form isolated from a living body, specifically, a hybridoma, which is capable of producing an antibody of the present invention, and is isolated from a living body. Examples include spleen cells, lymphocytes, transformed cells and the like. Any of these isolated cells can be used for producing the antibody of the present invention, and such cells as hybridomas are particularly useful for producing a monoclonal antibody which is a form of the antibody of the present invention. .
  • the spleen is excised from the immunized animal, dispersed, and spleen cells are collected. Obtained as antibody-producing cells. Splenocytes can also be further immunized in vitro, if desired. The spleen cells thus obtained are then fused with infinitely proliferable cells of warm-blooded animal origin.
  • Examples of cells capable of infinite proliferation include, for example, SP2 / 0-Ag14 cells (ATCCCRL-15881), Y3-Ag1.2.3 (ATCCCRL-16631), P 3 / NS 1/1-Ag 4-1 cells (ATCCTIB-18) and P3X63Ag 8 cells (ATCCT! B-9), etc., or cell lines derived from mouse or rat myeloma Such mutants can be mentioned, and more suitable ones are selected in consideration of the compatibility with the above spleen cells.
  • a conventional method using a fusion promoter such as polyethylene glycol-Sendai virus or an electric pulse is appropriately employed.
  • the cell fusion product is cultured in a selection medium such as a HAT medium according to a conventional method, and the fused cells, that is, hybridomas, are selectively grown.
  • a selection medium such as a HAT medium
  • the fused cells that is, hybridomas
  • a hybridoma according to the present invention is obtained. If a conventional method such as limiting dilution is applied to the selected hybrid doroma, the desired hybrid A redoma clone is obtained.
  • the eightybridoma thus obtained as a clone is cultured in vivo or in vitro and, if necessary, the desired immunoglobulin is collected in the body fluid or culture.
  • Application of the method results in a monoclonal antibody according to the present invention that has been purified to the desired level.
  • the monoclonal antibody of the present invention also includes a so-called “humanized antibody” which is usually prepared by a protein engineering technique.
  • a humanized antibody for example, mRNA is collected from a mammalian hybridoma obtained as described above, reacted with reverse transcriptase to produce cDNA, amplified by PCR, and then cloned. Then, the nucleotide sequences of the heavy and light chains in the monoclonal antibody of the present invention, in particular, the nucleotide sequences of the variable regions in the heavy and light chains, respectively, are determined. Next, a chimeric gene encoding a polypeptide obtained by fusing those variable regions with the constant region of a human antibody is prepared.
  • this chimeric gene When this chimeric gene is appropriately expressed in a host, it produces a monoclonal antibody that exhibits the same binding characteristics as the original monoclonal antibody, but has significantly reduced antigenicity to humans.
  • a method of humanizing a mammal-derived antibody is known in the art. For example, the method described in S. Paul, “Methods in Molecular Biology”, Vol. 51, 1995, Hyumana 'The press publication describes various techniques involved.
  • the antibody of the present invention including the polyclonal antibody and the form as a monoclonal antibody described above is obtained as a sample purified to a desired level by a conventional purification method in the art for purifying antibodies in general. be able to.
  • Individual purification methods For example, salting out, dialysis, filtration, concentration, centrifugation, fractional precipitation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography, gel electrophoresis Electrophoresis and isoelectric focusing, which are used in an appropriate combination as necessary.
  • the purified antibody is then concentrated and dried, depending on the application, to be in a liquid or solid form.
  • the peptide fragment referred to in the present invention is a part of the polypeptide of the present invention.
  • a polypeptide having an amino acid sequence represented by any one of SEQ ID NOs: 4 to 10 in the sequence listing preferably It is said that the polypeptide having a partial amino acid sequence represented by any one of SEQ ID NOs: 1 to 3 or a mutant thereof in the sequence listing is sufficient for an antibody to recognize as an antigenic determinant.
  • it means a peptide fragment having a continuous partial amino acid sequence of at least 10 residues, preferably at least 20 residues.
  • the peptide fragment of the present invention may be a polypeptide fragment artificially expressed by natural or recombinant DNA technology, partially degraded by an acid or a polypeptide degrading enzyme such as various proteases, etc. It may be an artificially synthesized one or an artificially expressed and prepared one by recombinant DNA branching.
  • the peptide fragment thus obtained is used in the same manner as the polypeptide of the present invention as long as it substantially enhances the expression of gelatinase in mammalian skin cells including humans or promotes the growth of hematopoietic cells. be able to. Even when the peptide fragment does not have the desired biological action, it can be used as an antigen for producing an antibody against the polypeptide of the present invention.
  • the polypeptide of the present invention has an activity of enhancing gelatinase expression in mammalian skin cells, in the field of pharmaceuticals, it can be used as a healing promoter when treating wounds in the skin or inflammation. It is useful for tissue reconstruction in regenerative medicine. Furthermore, it can be used in cosmetic surgery to reduce keloid scars.
  • wounds such as cuts and abrasions, skin damage due to burns, keloids, pressure ulcers, atopic dermatitis, and contact skin Inflammation, self-sensitizing dermatitis, seborrheic dermatitis, mouth dermatitis, exfoliative dermatitis (erythroderma), senile xeroderma, localized scleroderma, monetary eczema, juniper, These include hypertrophic scars, dandruff, acne, freckles and hot flashes.
  • it can be used for hair cosmetics, basic cosmetics, and the like.
  • the polypeptide of the present invention since the polypeptide of the present invention has an action of promoting the proliferation of hematopoietic cells in mammals, in the field of pharmaceuticals, it is necessary to treat hematological diseases such as aplastic anemia and the like when treating malignant tumors by chemotherapy and radiation therapy. It is useful as a hematopoietic cell proliferation promoter at the time of bone marrow transplantation for the treatment of immune diseases such as severe combined immunodeficiency disease, and at the time of expansion of hematopoietic cells for bone marrow transplantation.
  • the effects of the polypeptide of the present invention include renal anemia, pernicious anemia, aplastic anemia, myelodysplastic syndrome, chronic myeloid leukemia, chronic lymphocytic leukemia, and adults. T cell leukemia and the like. In the field of research dealing with hematopoietic cells, it can also be used as a research reagent for amplifying hematopoietic cells. Since the polypeptide of the present invention is originally derived from mammals, it is a safe substance with extremely low toxicity.
  • the antibody of the present invention specifically binds to the polypeptide of the present invention, it can be used for purification of the polypeptide of the present invention or qualitatively or quantitatively detecting the polypeptide of the present invention. It is extremely useful in fluorescence immunoassays and enzyme immunoassays.
  • the polypeptide of the present invention when used as a gelatinase expression enhancer in skin cells, it can be used for the purpose of suppressing overexpression of gelatinase. Furthermore, it can be used for the purpose of diagnosing or treating diseases and diseases associated with the overexpression of gelatinase.
  • the polypeptide of the present invention when used as an agent for promoting the proliferation of hematopoietic cells, it can be used for the purpose of suppressing the hyperproliferation of hematopoietic cells. It can also be used for diagnostic or therapeutic purposes.
  • the polypeptide of the present invention can be used alone, it can also be used in the form of a composition containing other components.
  • the composition comprising the polypeptide of the present invention can be advantageously used as cosmetics, pharmaceuticals and reagents. If necessary, the composition of the present invention may contain components other than the polypeptide of the present invention, i.e., components that are applicable to cosmetics and pharmaceuticals for mammals including humans.
  • composition of the present invention Used, for example, water, alcohol, starchy, protein, amino acids, fibrous, carbohydrates, lipids, fatty acids, vitamins, minerals, flavors, colorings, sweeteners, seasonings, spices, stabilizers, preservatives. It can also be advantageously carried out to include one or more components such as emulsifiers, emulsifiers, surfactants, excipients, extenders, thickeners and preservatives. These components are usually appropriately selected according to the needs in each field of application of the composition of the present invention.
  • Form of the composition of the present invention containing the above components There are no particular restrictions on the formulation, and it is provided in a desired form such as powder, granules, tablets, pastes, jellies, emulsions and solutions.
  • carbohydrate examples include sugars such as glucose, fructose, lactose, trehalose, maltose, sucrose, lactose, syrup, cyclic sugars such as cyclodextrin and cyclic tetrasaccharide, and erythri.
  • sugar alcohols such as tall, mannitol, sorbitol, xylitol, maltitol, reduced starch syrup, natural polysaccharides such as pullulan, carrageenan, natural gums, and carboxymethyl cellulose
  • the polypeptide of the present invention is prepared according to an appropriate composition selected according to the target animal or its administration method. As indicated, one or more components approved for use in either the cosmetics or pharmaceutical fields are mixed, diluted, and concentrated according to the purpose based on the individual compounding amount. Steps such as drying, filtration, and centrifugation are appropriately performed to prepare a composition containing the polypeptide, and the composition may be formed into a desired shape as needed. There are no particular restrictions on the order in which the components are blended or when each of the above steps is carried out, as long as the desired biological effect of the polypeptide is not reduced, and any of the above may be used as necessary. These steps may be performed singly or in combination as appropriate.
  • the polypeptide of the present invention is used for mammalian skin cells.
  • the composition comprising the polypeptide of the present invention has the effect of enhancing the expression of gelatinase and promoting the growth of hematopoietic cells of mammals or mammals. It can be advantageously used in applications requiring a substance having such an action in the field described above.
  • the composition of the present invention exhibits a marked gelatinase expression enhancing effect as the content of the polypeptide as an active ingredient increases.
  • the polypeptide may be highly purified or partially purified, in order to obtain a composition having a remarkable gelatinase expression enhancing action, as described in Examples below.
  • the relative expression of gelatinase was increased by at least 1.2 times when the polypeptide at a concentration of 10 ⁇ g ZmI was used compared to the case without the polypeptide. It is desirable to increase the content of the polypeptide to a level that allows it.
  • the composition of the present invention exerts a marked gelatinase expression enhancing effect when used transdermally.
  • the effective dose of the composition of the present invention varies depending on the type, age, sex, etc. of the mammal including the target human, but the weight of the polypeptide as the active ingredient in the composition of the present invention is not limited. In terms of conversion, per adult, usually 1 to 1 OOO g / time, desirably 10 to 500 g / time, once or several times a day, depending on the symptoms and dosage form, daily or It should be given at intervals of ⁇ days or more.
  • the administration form is not particularly limited, and transdermal or transmucosal, and in some cases, oral or tube administration may be appropriately selected and used.
  • a composition comprising the polypeptide of the present invention is When applied directly to the skin as a skin external preparation such as a product, the amount of polypeptide, which is the active ingredient in the composition of the present invention, is 0.0000 in weight of the total external preparation for skin. From 10 to 10% by mass, preferably from 0.000% to 1% by mass, depending on the effect once or several times a day. It can be applied to the skin. If the content is less than 0.001% by mass, the effect is difficult to be exhibited, and if the content exceeds 10% by mass, the further effect cannot be expected, which is not preferable.
  • Cosmetic products using the composition of the present invention as a gelatinase expression enhancer include, for example, lotions, creams, emulsions, gels, powders, pastes, blocks and the like. Soap, toilet soap, skin wash powder, face wash cream, face wash foam, facial rinse, body shampoo, body rinse, shampoo, rinse, hair wash powder, etc.
  • Cleansing cosmetics set-up lotions, hair blows, chips, hair creams, pommer hair sprays, hair liquids, hair tonics, hair rosins, hair tonics, hair dyes, Treatment for scalp, bottled oil, polish> shine, i> shine, hair cosmetics such as skim oil, lotion, vanishing cream, emollient cream , Emollient Cosmetics, packing cosmetics (such as jelly-like peel-off type, jelly-like wipe-off type, paste-type wash-down type, powder type, etc.), cleaning cream, cold storage Realms, Hand Creams, Hand Lotions, Emulsions, Moisturizers, After Shaving Lotions, Shaving Lotions, Press Lotions Products such as blow-on, after-shake cream, after-shake cream, pre-seam cream, cosmetic oil, baby oil, etc.
  • Basic cosmetics foundations (liquid, cream Reamed, solid, etc.), talcum powder, baby powder, body powder, perfume powder, make-up base, interesting (cream, paste, liquid, solid, powder, etc.), aishadow, Make-up cosmetics such as eye cream, mascara, eyebrows, eyelash cosmetics, lipstick, lipstick, lotion, etc., aromatic cosmetics such as perfume, condensed perfume, powdered perfume, cologne, perfume colon, eau de toilette, suntan Tanning, sunscreen lotion, tanning oil, sunscreen cream, sunscreen lotion, sunscreen oil, etc.
  • Nail cosmetics such as nail cream, nail cosmetics, eyeliner makeup , Lipstick, Li Ppuku stream, Neribeni, mouth lips cosmetics such as Li Ppudarosu, toothpastes, mouth, such as Mausuwosshu cosmetics, bus Sol Bok, bath oils, such as bathing cosmetics such as bath cosmetics can be mentioned up.
  • composition of the present invention can also be used as an agent for promoting the growth of hematopoietic cells derived from mammals.
  • an agent for promoting the growth of hematopoietic cells derived from mammals When used for the purpose of proliferating hematopoietic cells in vitro, it may vary depending on the origin of the polypeptide of the present invention and the origin of the hematopoietic cells to be applied.
  • Weight of the port re peptide is active ingredient, 1 X 1 0 5 per hematopoietic cells, usually, 0. 1 ig or more, is desirable and rather, contained in the culture solution or more 1 mu g Then, hematopoietic cells can be cultured and expanded.
  • the form of the agent it can be used in the same form as the above-mentioned gelatinase expression enhancer as long as the purpose of use as a hematopoietic cell proliferation promoter is not hindered.
  • the target product is produced at an appropriate time during the process of producing the target product according to a conventional production method. What is necessary is just to add the polypeptide of this invention. Although there is no particular limitation on the timing of addition, if the target product is manufactured through a heating step, add it after cooling down to room temperature, preferably 30 ° C or less, after the heating step. This makes it possible to prevent the expected biological action of the polypeptide of the present invention from attenuating in the production process.
  • the composition of the present invention as described above contains the polypeptide of the present invention in an amount of usually 0.01% by mass or more, preferably from 0.01 to 100% by mass, based on the weight of the product. I do.
  • the composition containing the polypeptide of the present invention as an active ingredient exhibits a gelatinase expression-enhancing effect on mammalian skin cells.
  • the effect of enhancing the expression of gelatinase is effectively exerted, and early alleviation and treatment of the wound can be achieved without causing serious side effects.
  • the composition of the present invention since the composition of the present invention has an action of promoting the growth of mammalian hematopoietic cells, it can be used as a research reagent or for amplifying bone marrow-derived hematopoietic cells in vitro for bone marrow transplantation and the like. It is useful as a hematopoietic cell proliferation promoter.
  • a composition containing an antibody against the polypeptide of the present invention as an active ingredient can also be prepared in the same manner as in the case of the polypeptide of the present invention.
  • a humanized antibody against a peptide, a chimera antibody, or a composition containing a human antibody as an active ingredient is used in a medical field
  • the polypeptide of the present invention in a human may be used.
  • the amount of the composition that is effective in treating a disease associated with overexpression of the peptide is usually selected based on the polypeptide level of the present invention in the human body.
  • the polypeptide level of the present invention in the body can be determined, for example, by immunologically using a biological sample such as a body fluid collected from a patient, for example, using a mouse antibody against the polypeptide of the present invention. It can be measured using a detection method. Comparison of the measured value with a reference value in a healthy person, which is measured in the same manner, suggests an excess amount of the polypeptide of the present invention in such a patient.
  • a composition containing the antibody of the present invention in an amount capable of neutralizing this excess amount in the body of the patient may be administered to the patient.
  • the amount of the antibody that can be neutralized varies depending on the form of the composition and the administration route, it is usually 1 to 2 or more in a molar ratio to the amount of the polypeptide of the present invention.
  • the dose of the composition selected in this manner may be administered once or in two or more doses in consideration of the type, symptom, and site of the disease. About 1 ⁇ g to 1 g per adult, more preferably 1 to 4 times / day or 1 to 5 times / week at a dose of about 10 g to 100 mg / time O
  • the antibody of the present invention suppresses the growth of vascular endothelial cells and / or the formation of sebocytes, as shown in the Examples below.
  • a susceptible drug containing a certain antibody as an active ingredient suppresses angiogenesis It is useful as an agent or a sebum production inhibitor.
  • the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited by these examples.
  • the primary PCR was carried out according to a conventional method using the above method. Next, a part of the reaction solution containing the primary PCR product obtained by amplification was designated as type III, and represented by SEQ ID NO: 21 or 22 in the sequence listing, and SEQ ID NO: 23 in the sequence listing. Secondary PCR was performed in the same manner by using a synthetic DNA having the base sequence to be used as a PCR primer.
  • a PCR primer having a base sequence represented by SEQ ID NO: 18 in the sequence listing was used,
  • a PCR primer having the nucleotide sequence shown in SEQ ID NO: 21 in R in R two types of PCR products having sizes of 0.5 kbp and 1.5 kbp were obtained.
  • R27L the one with 0.5 kbp was named R27L and the one with 1.5 kbp was named R27H.
  • PCR primer having the nucleotide sequence of SEQ ID NO: 19 in the sequence listing was used
  • secondary PCR a PCR primer having the nucleotide sequence of SEQ ID NO: 22 in the sequence listing was used From PCR amplification, various PCR products having a size of 0.3 kbp were obtained and named R23.
  • the amplified fragments R23, R27, and R27H obtained by PCR were purified by polyethylene glycol precipitation, respectively, and then the plasmid vector pBIuescript SK (digested with the restriction enzyme Ec0RV) was used.
  • -) (Stratagene) and DNA Ligation Shot Kit (2) (Yukara Bio Inc.) using the conventional method. Then, using the resulting recombinant plasmid E. coli Konbiten voxel - was (XL 1 0 G o I d K an r, manufactured by scan Bok Ratajin) was transformed.
  • the obtained transformant is inoculated into an L-broth medium ( ⁇ 7.2), cultured with shaking at 37 ° C for 18 hours, and the transformant is collected from the culture and subjected to a conventional alkaline method.
  • Plasmid DNA was prepared by one SDS method, and three types of transformants each containing the target PCR amplified fragment were selected.
  • Plasmid DNA was prepared from the three transformants obtained in the same manner as described above, PCR amplified fragments R23, R27 were prepared, and the nucleotide sequence of R27H was sequenced using a DNA sequencer (model 373A). , Applied Biosystems Japan Co., Ltd.) revealed that R23 and R27L or R23 and R27H had partially overlapping base sequences, and each had a partial base of cDNA. It turned out to be an array. In order to confirm that each is derived from one mRNA, the sequence number in the sequence listing as a forward primer was determined based on the base sequence near the predicted base sequence of the R23 initiation codon.
  • a DNA sequence having the nucleotide sequence represented by 24 and a sequence primer as a complementary strand primer based on the sequence near the sequence predicted to be the stop codon of R27L or R27H DNA having the nucleotide sequence of SEQ ID NO: 25 or SEQ ID NO: 26 was synthesized.
  • a reaction solution obtained by performing primary PCR using a DNA having the base sequence represented by SEQ ID NO: 18 in the sequence listing as a primer was designated as ⁇ , and the base sequence represented by SEQ ID NO: 24 and SEQ ID NO: 25 in the sequence listing was obtained.
  • Secondary PCR was performed again using a primer having the sequence or a combination of primers having the nucleotide sequences of SEQ ID NOs: 24 and 26 in the sequence listing.
  • a PCR product of 0.5 kb was obtained by PCR using a primer having a base sequence represented by SEQ ID NO: 24 or SEQ ID NO: 25 in the sequence listing, and designated as R48.
  • a PCR product of 0.6 kbp was obtained by PCR using a combination of the primers having the nucleotide sequences of SEQ ID NO: 24 and SEQ ID NO: 26 in the sequence listing, and was named R50.
  • PCR amplified fragments R48 and R50 were cloned into plasmid vector ⁇ Iuescript SK (-) j digested with restriction enzyme Ec0RV as above, and the nucleotide sequence was analyzed.
  • R48 was a partial nucleotide sequence of cDNA composed of R23 and R27L.
  • R 5 0 was a partial nucleotide sequence of cDNA composed of R23 and R27H.
  • Example 11-11 From the results of Example 11-11, it was found that R23 and R27L, and R23 and R27H were each derived from the same mRNA.
  • the cDNA composed of R23 and R27 is represented by SEQ ID NO: ⁇ ⁇ ⁇ ⁇ in the sequence listing, and the cDNA composed of R23 and R27H is represented by SEQ ID NO: 12 in the sequence listing. It had the nucleotide sequence shown.
  • the nucleotide sequence represented by SEQ ID NO: 11 in the sequence listing is the amino acid sequence consisting of 155 residues described in addition to the nucleotide sequence represented by SEQ ID NO: 11 in the sequence listing, and SEQ ID NO: 12 in the sequence listing.
  • the Kerachi Nosai Bok fraction of epidermal cells prepared from 4-day old hamsters dorsal skin, and cultured for 2 days at 3 7 ° C, 5% under the conditions of C 0 2 in keratinocytes Bok medium. Collect the cells and remove 1 to 4 X 1 0 were immunized intraperitoneally into BALB Bruno c mice 6 cells / animal. After this procedure was performed three times at 2-week intervals, blood was collected from the tail vein, and the serum was separated and used for antibody titer measurement. Further cell fusion 3 days and final immunization intraperitoneally with 2 X 1 0 7 cells / animal of keratinocytes Bok fraction recovered from 4-day-old hamsters skin as well.
  • Antibody titer measurement and screening were performed using the same preparation of the Hamus Yuichi keratinocyte fraction and 88/8/3/3 cells in a 96-well plate, followed by 4% paraformaldehyde.
  • CELLEIA method using cells immobilized with CELL as an antigen was used.
  • spleen was excised from a mouse with a serum showing a strong reactivity to keratinocytes in particular, and the spleen cell was fused with mouse myeloma cell SP2 / 0 by a conventional method.
  • Clones were selected by the method described above, and a monoclonal antibody-producing clone against the desired hamster hair follicle constituting cells was obtained.
  • the clone was named mAbK1141-1.
  • RNA was prepared from the cultured epidermis cells using RN easy Midik Mt (Qiagen Co., Ltd.). Next, the obtained total RNA was purified using an mRNA purification kit (Oligotex-dT30 ⁇ Super>, manufactured by Takara Labsai Co., Ltd.) and an ordinary method to prepare mRNA.
  • cDNA was prepared using reverse transcriptase (manufactured by Invitrogen Co., Ltd., trade name: “Super Script II j”) and random primer (manufactured by Invitrogen Co., Ltd.). Synthesize was. Furthermore, after adding a restriction enzyme BstXI adapter (manufactured by Invitrogen Co., Ltd.) to the synthesized cDNA, 1% agarose gel electrophoresis was performed to recover cDNA of 1 kbp or more.
  • the obtained cDNA was ligated with a plasmid vector pcDNAI / Amp (manufactured by Invitrogen Co., Ltd.) previously digested with BstXI using T4 DNA ligase, and then Electro MAXDH10 BT 2.5 kV, 25 AC F, 100 ⁇ using Gene Pulser Unit (manufactured by Nippon Marsh * Rad Laboratories Co., Ltd.) for 1 phageresistantce II s (manufactured by Invitrogen Corporation) It was introduced by the electroporation method under the following conditions. As a result 5.1 was obtained XI 0 5 transformants.
  • the antibody was removed by centrifugal washing. Next, the cells to which the antibody was bound were seeded on a 6 cm dish coated with a goat anti-mouse IgG antibody (manufactured by Jackson Imno Research, 10 tg / mI), and subjected to adsorption treatment at room temperature for 3 hours. Was. After washing off the non-adsorbed cells, the remaining adsorbed cells are dissolved in a heart solution (0.6% SDS, 1 Om MEDTA), and a 1 M final sodium chloride aqueous solution is added, and the mixture is added at 4 ° C. ⁇ ⁇ I left it. This was centrifuged at 15,000 rpm for 10 minutes, and the supernatant was recovered.
  • a goat anti-mouse IgG antibody manufactured by Jackson Imno Research, 10 tg / mI
  • Plasmid DNA of a single clone was prepared from the DNA containing the target gene, which was concentrated by the Banning operation. The obtained DNA was transfected into COS-1 cells using a gene transfer agent for Lipofexion (trade name: Lipofectamine 2000, manufactured by Invitrogen Co., Ltd.).
  • the cells were cultured on Dulbecco's modified Eagle's medium (D-MEM medium, manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10% fetal calf serum (FCS). After 2-3 days of culture, the cells are fixed with 2% paraformaldehyde, and a phosphate buffer containing 5% FCS containing 50 / mI monoclonal antibody mAb K114-I "I is added. After washing with a phosphate buffer, the mixture was reacted with an alkaline phosphatase-labeled anti-mouse IgG antibody (manufactured by Sigma-Aldrich Corp.) for 1 hour at room temperature. After washing with a phosphate buffer, the cells were stained with Immuno Pure Fast Red TR / AS-MXS ubstrate Kit (manufactured by Pierce) to obtain stained positive transformants.
  • D-MEM medium Dulbecco's modified Eagle's medium, manufactured by Nissui Pharmaceutical Co
  • Plasmid DNA was prepared from the obtained positive transformants in the same manner as above, and named pcD-hamAgKI14.
  • the nucleotide sequence of the inserted cDNA was prayed using a DNA sequencer (Model 373A, manufactured by Applied By Saisei Systems Japan K.K.).
  • the open reading frame had an amino acid sequence consisting of 2422 residues, which was added to the nucleotide sequence shown in SEQ ID NO: 13 in the sequence listing.
  • the polypeptide having this amino acid sequence was designated as hamAgKI14-1.
  • ham Ag KI 14 — 1 The amino acid residues 225 to 241 of ham Ag KI 14 — 1 are predicted to form a transmembrane region from the characteristics of the amino acid sequence, and ham Ag KI 14 — 1 is glycosylphosphatidylinositol (GPI) anchor type 1 membrane protein Presumed to be white matter.
  • GPI glycosylphosphatidylinositol
  • RNA was prepared from the cells using RN easy Midikit (manufactured by Qiagen). Next, 2 g of the obtained total RNA was designated as type II, and a reverse transcriptase (manufactured by Invitrogen Co., Ltd., trade name "Supper Scrip II") and a base represented by SEQ ID NO: 28 in the sequence listing were prepared. CDNA was synthesized using a primer having a sequence, dT anchor-primer. ⁇ Example 3-3: Cloning of DNA encoding mouse-derived polypeptide>
  • the cDNA synthesized above was designated as type II, and a combination of a forward-strand primer having a base sequence represented by SEQ ID NO: 27 in the sequence listing and a PCR anchor primer represented by SEQ ID NO: 29 in the sequence listing, PCR was performed up to the poly (A) sequence by a conventional 3 'RACE method using a DNA polymerase (Yukara Bio Inc., trade name "TaKaRaExtaqj"). 30 cycles were performed at 3 ° C, 30 seconds—60 ° C, 45 seconds ⁇ 72 ° C, 3 minutes This PCR resulted in a 1.3 kbp PCR amplified product (PCR13 and PCR13).
  • the resulting PCR13 was purified by the polyethylene glycol precipitation method, and the DNA was added to a plasmid vector pT7BIue (manufactured by Merck) digested with the restriction enzyme Ec0RV. Using Ligation Kit ver. 2 (manufactured by Taka Labay Sai Co., Ltd.), recombine in a conventional manner and use recombinant plasmid. (PTB—mAgK114-PCR13) was used to transform the E. coli recombinant cells (JM ⁇ 09, manufactured by Takara Bio Inc.) using the obtained recombinant plasmid.
  • Plasmid DNA is prepared by the usual alkaline SDS method, and a clone containing the target PCR amplified fragment is selected. I chose.
  • Plasmid DNA was prepared from the obtained transformant in the same manner as described above, and the base sequence of the PCR-amplified fragment PCR13 was sequenced using a DNA sequencer (Model 373A, manufactured by Applied By Saisei Systems Japan Co., Ltd.). As a result, it was found to have the nucleotide sequence represented by SEQ ID NO: 15 in the sequence listing.
  • This nucleotide sequence has an open reading frame consisting of 714 bases, and encodes an amino acid sequence consisting of 237 residues described in addition to the nucleotide sequence represented by SEQ ID NO: 15 in the sequence listing.
  • This nucleotide sequence has an open reading frame consisting of 714 bases, and encodes an amino acid sequence consisting of 237 residues described in addition to the nucleotide sequence represented by SEQ ID NO: 15 in the sequence listing.
  • the homology between the cDNA having the nucleotide sequence of SEQ ID NO: 15 in the mouse-derived sequence listing and the cDNA having the nucleotide sequence of SEQ ID NO: 13 derived from the hamster obtained in Example 2 is 75% Met.
  • the amino acid sequence described in SEQ ID NO: 15 in the sequence listing encoded by the cDNA and the amino acid sequence of a hamster-derived polypeptide (also described in the sequence listing as nucleotide sequence shown in SEQ ID NO: 13 in the sequence listing) The amino acid sequence) was 60%.
  • the amino acid sequence of the 220 to 226 residue of the mouse-derived polypeptide is predicted to form a transmembrane region like the hamster-derived polypeptide, and the mouse-derived polypeptide also has glycosylphosphatidylinos. It was estimated to be a Thor (GPI) anchor type 1 membrane protein.
  • GPI Thor anchor type 1 membrane protein.
  • a database search was performed based on the amino acid sequence described in SEQ ID NO: 15 in the sequence listing of the mouse-derived polypeptide, and the result was obtained.
  • DNA code Completely matched the amino acid sequence of a polypeptide of unknown function.
  • the PCR product amplified by the 3 ′ RACE method in Example 3 was subjected to 2% agarose electrophoresis in order to clone a smaller secretory polypeptide, and the DNA band at a position corresponding to about 800 bp ( PCR 181) and extracted using a DNA extraction kit (Qiagen Co., Ltd., trade name “QIAEXI 1”) to purify the DNA.
  • PCR 181 was digested with the restriction enzyme Ec0RV into a plasmid vector pT7BIue (manufactured by Merck Ltd.) to obtain a DNA ligation kit Ver.2.
  • Example 4-12 Determination of base sequence and encoded amino acid sequence> Plasmid DNA was prepared from the obtained transformant in the same manner as described above, and the base sequence of the PCR-amplified fragment PCR181 was sequenced using a DNA sequencer (Model 373A, Applied Dubai Systems Japan Ltd.). As a result, it was found to have the nucleotide sequence represented by SEQ ID NO: 17 in the sequence listing.
  • the nucleotide sequence represented by SEQ ID NO: ⁇ 7 in the sequence listing has an open reading frame consisting of 585 bases, and has 194 residues described in addition to the nucleotide sequence represented by SEQ ID NO: 17 in the sequence listing.
  • amino acid sequence consisting of The homology with the cDNA having the nucleotide sequence of SEQ ID NO: 15 in the mouse-derived sequence listing obtained in Example 3 was 75%.
  • amino acid sequence described in SEQ ID NO: 17 in the sequence listing encoded by the cDNA and the amino acid sequence of the polypeptide of Example 3 shown as SEQ ID NO: 15 in the sequence listing
  • the amino acid sequence described with the base sequence described above was completely identical from residue 1 to residue 148, but the subsequent sequences were completely different. Since this polypeptide does not have a putative GPI-anchored transmembrane region on the amino acid sequence, it was considered to be a secretory polypeptide.
  • An Xh0I recognition sequence was prepared at the 5 'end of the cDNA having the nucleotide sequence represented by SEQ ID NO: 11 in the sequence listing, and the amino acid sequence represented by SEQ ID NO: 30 (FLAG sequence) in the sequence listing was produced at the 3' end. ), And 10 ng of plasmid DNA into which cDNA having the nucleotide sequence shown in SEQ ID NO: 11 in the sequence listing was inserted, for the purpose of preparing a nucleotide sequence encoding N0tI recognition sequence.
  • PCR was performed using a combination of a forward-strand primer having the base sequence of SEQ ID NO: 31 in the sequence listing and a complementary primer having the base sequence of SEQ ID NO: 32 in the sequence listing.
  • the amplified fragment obtained was purified by the polyethylene glycol precipitation method, and then purified by the SrfI site of plasmid (“pCR—Script Cam SK (10)” manufactured by Stratagene Co., Ltd.). Was cloned.
  • plasmid having an Xh0I recognition sequence at the 5 'end, a nucleotide sequence encoding the FLAG sequence at the 3' end, and a NotI recognition sequence was as planned. Obtained.
  • the obtained plasmid DNA was digested with Xh0I and N0tI to prepare an Xh0I-otI fragment containing cDNA having a base sequence represented by SEQ ID NO: 1 in the sequence listing.
  • the expression vector pCDM8 (manufactured by Invitrogen Co., Ltd.) was inserted into the XhoI-NotI site downstream of the CMV promoter.
  • the obtained expression vector was designated as pcD-hAgK114aFL.
  • the polypeptide expressed using this expression vector is a fusion polypeptide having a FLAG sequence at the C-terminal position of the polypeptide having the amino acid sequence described in the base sequence of SEQ ID NO: 11 in the sequence listing. Is obtained as Figure 1 shows the basic structure of the constructed expression vector pcD—hAgK114aFL.
  • the expression vector pcD-hAgK114aFL obtained in Example 5-1 was introduced into COS-1 cells by the lipofection method as follows to transform the cells.
  • 10 UL g of plasmid DNA was diluted to 1.5 ml with D-MEM medium, and a lipofection method gene transfer reagent (trade name “ribofectamine 2000”, invitrogen) was used.
  • Rogen Corp. hereinafter abbreviated as LF200.
  • 60 fxI is diluted with D-MEM medium to 1.5 ml and left at room temperature for 5 minutes. The reaction was carried out for 0 minutes to form a DNA-LF20000 complex.
  • a culture solution of COS-1 cells was inoculated on a 10 cm plastic Petri dish with 4.8 ⁇ 10 6 ZL 0 ml of D-MEM medium containing 10% FCS and cultured overnight. The supernatant was removed, 5 ml of D-MEM medium was added, and 3 ml of DNA-LF20000 complex was added. After culturing the cells at 37 ° C. for 5 hours in the presence of 5% CO 2 , the supernatant was removed, the medium was replaced with 10 ml of D-MEM medium containing 10% FCS, and the cells were cultured again. After overnight culture, the medium was replaced with a serum-free medium for animal cells, ASF (manufactured by Ajinomoto Co., Inc.), at 5 ml, and the cells were further cultured for 3 days.
  • ASF manufactured by Ajinomoto Co., Inc.
  • the C-terminal of the polypeptide having the amino acid sequence described in SEQ ID NO: 11 in the sequence listing was added to the C-terminal.
  • Purification of the recombinant fusion polypeptide having the FLAG sequence was performed as follows. I went. The culture supernatant obtained by centrifuging the culture was centrifuged. The anti-FLAGM 2 was previously equilibrated with 5 O mM Tris-HCl buffer (pH 7.4) containing 0.1 M sodium chloride.
  • the antibody was applied to an antibody agarose column (volume: 5 ml, manufactured by Sigma-Aldrich Corporation) to specifically adsorb the recombinant fusion polypeptide having a FLAG sequence. After thoroughly washing with the same buffer to remove unadsorbed components, the adsorbed components were eluted using a 0.1 M glycine monohydrochloride buffer (pH 3.5). The polypeptide eluted fraction was neutralized by adding 1 M Tris-hydrochloric acid buffer (pH 8.0).
  • the recovered fraction is concentrated with an ultrafiltration membrane (trade name “Ultrafree”, manufactured by Millipore Co., Ltd.), and a gel with a volume of ⁇ 2 O ml pre-equilibrated with PBS (pH 7.1).
  • the product was applied to a filtration column (trade name “Superdex 200”, manufactured by Amersham Biosciences Inc.) and eluted with the same buffer.
  • the polypeptide was recovered using the absorbance at 280 ⁇ m as an index to obtain about 0.15 mg of the polypeptide in a yield of about 80%. This polypeptide was named hAgK114-4aFL.
  • Example 6 — 1 Preparation of recombinant baculovirus> Human-derived polypeptide hAgKI144-a cDNA encoding the FL and BD Pharmingen's BD Bakulogolod 'Transufexion' kit (BDB acu IoG old Transformation Kit) ” was used to prepare a recombinant baculovirus for polypeptide expression in insect cells.
  • PcR into which a DNA having an Xh0I recognition sequence at the 5 'end and a nucleotide sequence encoding a FLAG sequence and a N0tI recognition sequence at the 3' end obtained in Example 5-1 was inserted.
  • FIG. 2 shows the basic structure of the constructed expression vector pVL1393-hAgK114-4aFL.
  • a recombinant virus was produced using Sf9 insect cells (ATCCCRL-1711, derived from Orthodox moth) in accordance with the operation method described in the instructions attached to the kit.
  • Sf9 was seeded on a 6-well plate at 1 ⁇ 10 6 cells / well using TC 100 medium (manufactured by Invitrodin Co., Ltd.) supplemented with 10% (v Z v) FCS. After 10 minutes, the supernatant was removed, and then replaced with 0.5 ml of transfusion buffer A solution (Grace's medium containing 10% (V / V) FCS). Add 1.5 pVL1393-hAgK1141-aFL and 0.25; g BD Baculologo baculovirus DNA in advance and react for 5 minutes.
  • transfusion ⁇ / buffer B solution 1 A mixture containing 25 mM calcium chloride, 140 mM sodium chloride, 25 mM MHEPES, pH 7.1) was added at 0.5 ml / well, and the mixture was added at 27 ° C for 4 hours. Infected. Next, each well was washed once with 10% (V / V) FCS-supplemented TC-100 medium, 2 ml of the same medium was added, and the cells were further cultured at 27 ° C for 6 days. Was. Each culture was centrifuged at 1,000 rpm for 5 minutes to recover the supernatant, which was used as an hAgK114-1a FL recombinant baculovirus preparation.
  • S f 9 cells 1 X ⁇ 0 7 of the above preparation liquid 5 0 to 2 0 0 I was added to the infected one week at 2 7 ° C, and centrifuged The obtained supernatant was prepared as a recombinant virus solution for polypeptide expression.
  • Insect cell line “High Five” (manufactured by Invitrogen Co., Ltd., derived from Iraq Saginba) was used as a polypeptide expression cell.
  • Insect cell lines prepared L- glutamine (final concentration Im g / ml) with the added Express Five serum free medium (manufactured vivo Bok Rozhen (Ltd.)) ⁇ X 1 0 8 pieces / ⁇ 0 m I Then, 200 nI of the recombinant virus solution obtained in Example 6-1 was added, and 1
  • a recombinant fusion polypeptide having a FLAG sequence at the C-terminal portion was purified as follows.
  • the transfusion solution was centrifuged at 5,000 rpm for 45 minutes.
  • the anti-FLAGM2 antibody agarose column (5 ml I, Sigma-Aldrich) pre-equilibrated with 50 mM Tris-hydrochloric acid buffer (pH 7.4) containing 0.15 M sodium chloride (Manufactured by Tsuchi Corporation) to specifically adsorb the recombinant fusion polypeptide having a FLAG sequence.
  • the adsorbed components were eluted using 0.1 M glycine-hydrochloric acid buffer (PH 3.5).
  • the polypeptide eluted fraction was neutralized by adding 1 M Tris-HCl buffer (pH 8.0).
  • the recovered fraction is concentrated by an ultrafiltration membrane (trade name: Ultra-free, manufactured by Millipore Co., Ltd.) to recover the ribopolypeptide to obtain about 530 g of the polypeptide.
  • Ultra-free manufactured by Millipore Co., Ltd.
  • the 1st to 25th amino acid sequences are signal sequences for secretion, and the 26th and subsequent amino acid sequences.
  • PCR was performed using the cDNA-inserted plasmid DNA having the nucleotide sequence shown in SEQ ID NO: 12 in the sequence listing as type III.
  • An expression vector was obtained by operating in the same manner as in Example 5-1 except that the procedure was performed, and named pcD-hAgK114bFL.
  • the expressed polypeptide can be obtained as a fusion polypeptide having a FLAG sequence at the C-terminus of the polypeptide having the amino acid sequence described in SEQ ID NO: 12 in the sequence listing.
  • Figure 3 shows the basic structure of the constructed expression vector pcD—hAgK114bFL.
  • COS-1 cells were transformed in the same manner as in Example 5-2 except that pcD—hAgK114bFL obtained in Example 7-1 was used as an expression vector, and cell culture was performed. Was performed.
  • the recombinant fusion polypeptide having a FLAG sequence at the C-terminus was purified in the same manner as in Example 5-3. As a result, about 0.1 mg of the polypeptide was obtained in a yield of about 80%. Was. Using the obtained purified recombinant polypeptide, SDS-PAGE was carried out in the presence of a reducing agent according to a conventional method, and a single polypeptide at a position corresponding to a molecular weight of 30 to 100 kDa was obtained. Band detected. Common law As a result, when the N-terminal amino acid sequence of this purified polypeptide was examined at 5 residues, it was found to have the amino acid sequence represented by SEQ ID NO: 39 in the sequence listing.
  • amino acid sequence completely coincided with the 26th to 30th amino acid sequences of the amino acid sequence described in the nucleotide sequence shown in No. 12. From this, out of the amino acid sequences described together with the nucleotide sequence shown in SEQ ID NO: 12 in the sequence listing, the amino acid sequences from the 1st to the 25th are the signal sequences for secretion, and It was confirmed that the second and subsequent amino acid sequences, ie, the amino acid sequence represented by SEQ ID NO: 5 in the sequence listing, were the amino acid sequences of the mature polypeptide.
  • polypeptides having the amino acid sequences represented by SEQ ID NOs: 4 and 5 in the sequence listing have in common the partial amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, and this amino acid sequence has a biological effect. It was determined to be important.
  • Example 8
  • the cDNA having the nucleotide sequence represented by SEQ ID NO: 13 in the sequence listing cloned in Example 2 is a membrane-bound cDNA having the amino acid sequence described in addition to the nucleotide sequence represented by SEQ ID NO: 13 in the sequence listing.
  • hamster-derived polypeptide hamAgKI14-1 is coded, a C-terminal region predicted to be involved in membrane binding to obtain a secreted polypeptide (shown by SEQ ID NO: 13 in the sequence listing) In the amino acid sequence described along with the base sequence 22) The C-terminal region after the third serine residue) is deleted, and a recombinant fusion polypeptide having a FLAG sequence at the C-terminal portion is obtained in the same manner as in Examples 5 and 6. Thus, an expression vector having a modified cDNA was constructed.
  • Recombinant plasmid pcD—hamAgKI140140 ng obtained in Example 2 was designated as type I, and the synthetic DNA having the base sequence represented by SEQ ID NO: 34 in the sequence listing was used as a normal-chain primer. PCR was performed using a synthetic DNA having a base sequence represented by SEQ ID NO: 35 in the sequence listing as a complementary strand primer. The amplified fragment obtained is purified by the polyethylene glycol precipitation method, and then the plasmid vector pCR—ScriptCamSk (+)
  • the XhoI—NotI fragment containing the cDNA was excised again, and the expression vector pREF—XN (Taniguchi et al., Journal, Journal of Immunological Methods (J0 urna I of Immuno Iogica IM ethods)) , 1989, 2117, pp. 97-102) EF-1 downstream of the 1 ⁇ promoter,
  • the expression vector prepared by inserting into the XhoI-NotI site was named pRER-hamAgKI14d2FL.
  • the basic structure of the constructed expression vector pRER—hamAgKI14d2FL is shown in FIG.
  • Example 8-2 Transformation and culture of CHO—K1 cells>
  • the expression vector pRER-hamAgK114d2FL obtained in Example 8-1 was introduced into CH0-K1 cells by the electroporation method as follows to transform the cells. did. First, a positive Mi de DNA 1 O g, previously conventional methods CHO had been prepared by - mixing the cells 1 X 1 0 6 or containing liquid 8 0 0 ⁇ I in electronics Toropo Reshiyo emissions for cuvette in preparative And left at ice temperature for about 0 minutes. Subsequently, electrification was performed by applying an electric pulse of 25 At FD, 100 V twice twice at one-minute intervals.
  • One cell line was selected, single cloned by a conventional method, and named C ⁇ 0—hamAg ⁇ 114-1d2FL.
  • the cells were seeded on a 5% FCS-RPMI164 medium containing 34.5 mg / l L-proline and cultured at 37 ° C in the presence of 5% CO 2 . Cells are confluent After reaching the end state, the medium was replaced with ASF medium and cultured for 3 days.
  • the specific recombinant fusion polypeptide was adsorbed. After thoroughly washing with the same buffer to remove unadsorbed components, the adsorbed components were eluted using 0.1 M glycine-hydrochloric acid buffer (pH 3.5). The polypeptide eluted fraction was neutralized by adding 1 M Tris-HCl buffer (pH 8.0). The recovered fraction was concentrated with an ultrafiltration membrane (trade name: “Ultrafree”, manufactured by Millipore Co., Ltd.), and the volume of 120 m previously equilibrated with PBS (pH 7.1) The product was applied to a gel filtration column (trade name “Superdex 200”, manufactured by Amersham Baisai Science Co., Ltd.) and eluted with the same buffer. The polypeptide was recovered using the absorbance of 28 Onm as an index to obtain about 1.8 mg of HamAgKI14-1d2FL.
  • the cDNA having the nucleotide sequence of SEQ ID NO: 15 in the sequence listing cloned in Example 3 encodes a membrane-bound mouse-derived polypeptide mAgK114-1.
  • the C-terminal region involved in membrane binding (SEQ ID NO: 15 in the sequence listing) was used in the same manner as in the case of the hamster of Example 8.
  • the region at the C-terminal side after the 209th proline residue was deleted, and the recombinant having a FLAG sequence at the C-terminal was used in the same manner as in Examples 5, 7 and 8.
  • Recombinant plasmid pTB—mAgKl14 PCR obtained in Example 3 was used as the type III, and the synthetic DNA having the nucleotide sequence represented by SEQ ID NO: 36 in the sequence listing was sequenced. PCR was performed using a synthetic DNA having a base sequence represented by SEQ ID NO: 37 in the sequence listing as a complementary strand primer as a strand primer. The resulting amplified fragment was purified by a polyethylene glycol precipitation method, and then cloned into the SrfI site of plasmid vector pCR—ScriptCam SK (+) (Stratagene, Inc.).
  • the Xh0I recognition sequence at the 5 ′ end and the N0tI recognition sequence at the 3 ′ end of the nucleotide sequence represented by SEQ ID NO: 16 in the sequence listing were as planned. Each had an added nucleotide sequence. Since this DNA coded the amino acid sequence described in addition to the base sequence represented by SEQ ID NO: 16 in the sequence listing, it was originally intended to be the first to 218th mAg K114-1-1 as intended.
  • smAgK114-1FL cDNA encoding a recombinant secretory mutant fusion polypeptide having a FLAG sequence at the C-terminus of the amino acid sequence.
  • the Xh0I—NotI fragment containing the cDNA was excised again, and the expression vector p REF—XN downstream of the EF-1 ⁇ promoter, XhoI—N0t as in Example 8-1.
  • the expression vector prepared by insertion into the I site It was named pRER-smAgK114F.
  • Fig. 5 shows the basic structure of the constructed expression vector pRER-smAgK114FL.
  • CHO-smAgKI14FLBP23-1 One strain was selected, single cloned by a conventional method, and named CHO-smAgKI14FLBP23-1.
  • the cells 3 4. 5% FCS containing teeth one proline of 5 mg / l - plated in RPMI 1 6 4 0 medium and incubated at 3 7 ° C, 5% C 0 2 presence. After the cells became confluent, the medium was replaced with ASF medium and cultured for 3 days.
  • Example 9-13 Purification of polypeptide> CHO—sm Ag KI 14 FLBP 23 — “A mutant fusion poly- mer having the amino acid sequence described in SEQ ID NO: 16 in the sequence listing, using 1 cell culture solution 20 I as a purification material.
  • the adsorbed components were eluted using 0.1 M glycine monohydrochloride buffer (pH 3.5).
  • the peptide-eluting fraction was neutralized by adding 1 M Tris-monohydrochloride buffer (pH 8.0)
  • the recovered fraction was separated by ultrafiltration membrane (trade name “Ultrafree”, Millipore Co., Ltd.).
  • Gel filtration column (trade name "Superdex 200", manufactured by Amersham Biosciences, Inc.) preliminarily equilibrated with PBS (pH 7.1).
  • the polypeptide was recovered using the absorbance at 28 O nm as an index to obtain about 8.6 mg of smAgKI14-1FL.
  • the amino acid sequence up to the 26th position is a signal sequence for secretion, and the amino acid sequence at the 27th to 218th positions, that is, the amino acid sequence represented by SEQ ID NO: 9 in the sequence listing is that of the mature polypeptide. The amino acid sequence was confirmed.
  • Example 10 The amino acid sequence up to the 26th position is a signal sequence for secretion, and the amino acid sequence at the 27th to 218th positions, that is, the amino acid sequence represented by SEQ ID NO: 9 in the sequence listing is that of the mature polypeptide. The amino acid sequence was confirmed.
  • Recombinant plasmid pTB—mAgKI14PCR18 110 ⁇ ng obtained in Example 4 was designated as ⁇ , and the sequence number in the sequence listing was
  • PCR was performed using a synthetic DNA having a base sequence represented by 36 as a normal-strand primer and a synthetic DNA having a base sequence represented by SEQ ID NO: 38 in the sequence listing as a complementary-strand primer.
  • the obtained amplified fragment was purified by a polyethylene glycol precipitation method, and then cloned into the SrfI site of plasmid vector pCR—Script Cam SK (+) (Stratagene, Inc.).
  • the Xh0I recognition sequence at the 5 'end and the N0tI recognition sequence at the 3' end of the nucleotide sequence represented by SEQ ID NO: 17 in the sequence listing as planned. Have the added base sequences.
  • This DNA has the sequence Since the amino acid sequence represented by SEQ ID NO: 0 in the table was encoded, cDNA encoding a secreted polypeptide (named mAgK1 14—1b) was prepared. It was confirmed. The XhoI-NotI fragment containing the cDNA was excised again and inserted into the XhoI-Not1 site downstream of the EF-1 promoter of the expression vector PREF-XN in the same manner as in Example 8-1. The expression vector thus prepared was prepared using PRER-mAgK11.
  • Fig. 6 shows the basic structure of 14b.
  • Example 10 Expression vector obtained in 0-1 pRER-mAgK11
  • the adsorbed components were eluted using 0.4 MN-acetyldarcosamine monophosphate buffer (PH 7.4).
  • the polypeptide-eluting fraction was concentrated with an ultrafiltration membrane (trade name “Ultrafree”, manufactured by Millipore Co., Ltd.) and the volume of 5 O previously equilibrated with PBS (pH 7.5).
  • the mixture was applied to a column of ml of chelate (Cu2 + )-Sepharose (manufactured by Amersham Biosciences) and eluted with 2 OmM sodium phosphate buffer (pH 6.0).
  • fibroblasts (N) from normal human neonatal foreskin skin were placed in a 12-well microplate.
  • the cells were cultured for 2 days in the presence of 5% CO 2 gas. Then the proliferated N
  • Gelatinase activity was evaluated by the activity staining method. That is, each culture supernatant was subjected to electrophoresis using a polyacrylamide gel for activity staining containing 1 mg / ml of gelatin, followed by electrophoresis. Thereafter, the gel was treated with ⁇ in an incubation buffer to activate and react with lyserase. Next, the gel in the gel was stained with Coomassie Priliant Blue to detect a band that was whitened out due to gelatin degradation. The intensity of this band was measured all over the densitometer, and the activity of the test system for degrading gelatin was relatively evaluated using a control of ⁇ 100.
  • gelatinase A protein was evaluated by a conventional western blotting method. That is, the respective culture supernatants 12I were subjected to SDS-PAGE in the presence of a reducing agent by a conventional method using a gradient gel (manufactured by Daiichi Pure Chemicals Co., Ltd.) of 4% to 20% acrylamide. After that, the protein was transferred to a nitrocellulose membrane according to a conventional method. After blocking the transferred two-cellulose membrane using a blocking agent (trade name “PROC-ACE”, manufactured by Dainippon Pharmaceutical Co., Ltd.), ⁇ jag / ml anti-gelatinase A antibody is used as the primary antibody.
  • a blocking agent trade name “PROC-ACE”, manufactured by Dainippon Pharmaceutical Co., Ltd.
  • the recombinant polypeptide hAgKI14—1aFL derived from human was approximately three times as high as that without the control (control) at a concentration of 1 jug / mI.
  • the expression of gelatinase A protein in NHDF was remarkably enhanced by 9 times or more in a dose-dependent manner.
  • the recombinant polypeptide hAgK114--1aFL was almost the same as the control at a concentration of 1 ⁇ g / mI and 10 ⁇ g / ml.
  • fibroblasts derived from normal human neonatal foreskin skin, fibroblasts derived from hamster neonatal skin (FB) or mesenchymal cells derived from mouse fetal aorta (SC9-1) were placed in a 12-well microplate.
  • Gelatinase activity expressed in fibroblasts or mesenchymal cells was evaluated by the activity staining method used in Example 11. That is, each culture supernatant is subjected to electrophoresis using a polyacrylamide gel for active staining containing 1 mg / ml of gelatin, and after the electrophoresis, the gel is treated in an incubation buffer. In particular, the enzyme was activated and reacted. Next, the gelatin in the gel was stained with Coomassie Pleuriant Blue to detect bands that were whitened out due to gelatin degradation. The strength of the band was measured with a densitometer, and the activity of degrading gelatin in the test system was relatively evaluated using the control as ⁇ 100. The results are shown in Table 2. Table 2
  • the gelatinase activity evaluated by the activity staining method was higher than that of the FLAG peptide-added system (control) compared to the human-derived polypeptide hAgK114-1-1.
  • aFL and hAg ⁇ 1114- 1bFL can increase the gelatinase activity in NHDF by 1-5 times and 1.4 times, and increase the hamster-derived polypeptide hamAgK114-1-1d2FL. Increased the activity of gelatinase in FB by a factor of 1.24, and the mouse polypeptide smAgK
  • both the human-derived polypeptide hAg ⁇ 114-aFL and the ham-Ausu-ichi polypeptide hamAgK114-1d2FL are mouse-derived mesenchymal systems. Since the expression of gelatinase was weakly enhanced in cells SC9-19, these polypeptides were expected to exhibit the ability to enhance the expression of gelatinase across species. It was speculated. In this test, the recombinant polypeptide of the present invention showed gelatinase expression in fibroblasts or mesenchymal cells 1.2 times that of the control under the addition condition of a final concentration of 10 UL g / m ⁇ . The above was found to increase. Since the polypeptide of the present invention remarkably enhances the expression of gelatinase in fibroblasts or mesenchymal cells, it can be used to promote the healing of wounds and inflammation in the skin.
  • mice showed an effect of enhancing the expression of gelatinase, and the degree of the enhancement was about 1%. 7 times.
  • the mouse-derived polypeptide smAgKI1411FL-applied group had significantly smaller areas of both linear wounds and open wounds 7 days after wounding than the control group ( ⁇ serum albumin group). The values are shown.
  • no significant difference was observed in the expression of the gelatinase and in the wound area in the FLAG peptide application group as compared with the control group. It was determined that the mouse-derived polypeptide s mAg K I 14-1 FL enhanced the expression of gelatinase in mouse skin cells, and as a result, promoted the healing of skin wounds.
  • gelatinase A in the wound site of the mouse was evaluated by the following immunostaining method.
  • the epidermal tissue at the wound site of the mouse was cut out, fixed with 10% formalin, and then embedded with livalafin by a conventional method.
  • paraffin sections were prepared, deparaffinized with xylene, and washed with a water-alcohol mixture to gradually reduce the organic solvent concentration, and finally washed with PBS.
  • the endogenous peroxidase was deactivated by treatment with methanol containing 0.3% hydrogen peroxide, and then a blocking agent (trade name “Block Ace TM”, Dainippon Pharmaceutical Co., Ltd.) ) At room temperature for 30 minutes.
  • a mouse anti-human MMP-2 (gelatina-zeA) antibody (manufactured by Daiichi Fine Chemical Co., Ltd.) diluted 250-fold as a primary antibody was placed on the tissue section, and reacted at 4 ° C for 1 hour. .
  • the primary antibody was washed away with PBS, and then a goat anti-mouse immunoglobulin (trade name "EnVision + TM", manufactured by Dako's Japan Co., Ltd.) labeled with Perishin-Kisidase was used as the secondary antibody.
  • a goat anti-mouse immunoglobulin (trade name "EnVision + TM", manufactured by Dako's Japan Co., Ltd.) labeled with Perishin-Kisidase was used as the secondary antibody.
  • EndVision + TM manufactured by Dako's Japan Co., Ltd.
  • Bone marrow cells prepared from the femur of an adult hamster (8-week-old female) were adjusted to a concentration of 1 ⁇ 10 6 cells / ml using D-MEM medium containing 10 (v / v)% FCS. Then, the cells were inoculated in a 96-well microplate coated with collagen in an amount of 1 ⁇ m at a time. Next, the hamster-derived polypeptide hamAgK114-d2FL obtained by the method of Example 8 was similarly added to a D-MEM medium containing 10 (v / v)% FCS for 12.
  • the resulting solution was adjusted to a concentration of 100 ⁇ l, and 100 I was added thereto to make a total volume of 200 ⁇ I (the final concentration of the polypeptide was 50 ⁇ g / mI).
  • the expanded cells were fixed with 4% paraformaldehyde, treated with methanol according to a conventional method, and subjected to Giemsa staining. Then, these micrographs were taken, four fields were randomly selected, the number of cells was counted, and the average number of cells per field was calculated. Controls were performed in the same manner without adding the polypeptide, and the relative cell number when the polypeptide was used was expressed as a percentage, with the control cell number being 100%. Table 6 shows the results. Table 6
  • Bone marrow cells prepared from the femur of BALB mice were cultured at a concentration of 1 ⁇ 10 6 cells / m 1 in D-MEM medium containing 10 (V / V)% FCS. And then seeded on a 96-well microplate coated with collagen at a rate of 1001. Then, the Hams Yuichi-derived polypeptide hamAgK114-1d2FL obtained in Example 8 was similarly purified using a D-MEM medium containing 10 (v / v)% FCS. The solution was adjusted to a concentration of 5 Mg / mI, added with 100 ⁇ I, and used as a total solution I (the final concentration of the polypeptide was 12.5 Ag XmI).
  • BALBZ c mice (5 weeks old, female) 2 0 7. 5 X 1 0 5 cells using a D one MEM medium containing (V / V)% FCS bone marrow-derived cells, including hematopoietic cells prepared from the femoral bone of The mixture was adjusted to a concentration of / ml and inoculated into collagen-coated 8-well chamber slides (manufactured by Nalje Nunc International Co., Ltd.) at a ratio of 200 I. Next, a purified sample of the human-derived polypeptide hAgKl ⁇ 4-1a FL obtained by the method of Example 5 was similarly used to prepare a D-MEM medium containing 20 (V / V)% FCS.
  • Example 5 in saline containing 5% (w / w) gum arabic — An appropriate amount of the human-derived polypeptide obtained by the method of Example 3, the hamster-derived polypeptide obtained by the method of Example 3 or the mouse-derived polypeptide obtained by the method of Example 9-13 was dissolved. Thereafter, the bacteria were removed by filtration according to a conventional method. These were injected intraperitoneally into ddY mice weighing 20 to 25 g (group of 10 mice, Z) or orally by gastric tube, and the progress was observed for 7 days. As a result, no mortality was observed in any of the samples or any of the administration routes, even at the maximum dose of 20 mg / kg body weight. This result indicates that the polypeptide of the present invention is a safe substance that can be commonly used in mammals including humans.
  • Example 20 An appropriate amount of the human-derived polypeptide obtained by the method of Example 3, the hamster-derived polypeptide obtained by the method of Example 3 or the mouse-derived polypeptide obtained by the method of Example
  • Appropriate amounts of the human native polypeptide obtained in Example 5, the hamster-derived polypeptide obtained by the method of Example 8, or the mouse-derived polypeptide obtained by the method of Example 10 are dissolved in physiological saline, respectively. After that, 50 ⁇ l of the solution was dropped on a circular filter paper of a patch test patch (Taisho Pharmaceutical Co., Ltd.). These were applied to the skin of dd @ mice (10 mice / group) weighing 20 to 25 g, which had been depilated with a hair removal cream on the back in the usual manner, for 24 hours. Next, the stuck sample was removed, and after a lapse of 30 minutes, visual judgment was made to check for erythema, edema, papules and the like.
  • polypeptide of the present invention is a substance that is safe even if used regularly on the skin of mammals including humans.
  • J W ⁇ herons (female, weighing 2.5 k) were immunized according to the following schedule.
  • the recombinant polypeptide derived from the human hAgK114_1aFL obtained by the method of Example 5-3 together with complete Freund's adjuvant by the usual method was used as an antigen, and the initial immunization was carried out. It was injected subcutaneously at a dose of 0 g / animal. Every two weeks thereafter, as a booster, the same antigen was injected subcutaneously twice at the same dose together with incomplete Freund's adjuvant.
  • the polyclonal antibody of this example is extremely useful in the purification of the polypeptide of the present invention and the qualitative or quantitative detection of the polypeptide of the present invention, for example, in a fluorescent immunoassay, an enzyme immunoassay and the like. is there. Further, it is useful for suppressing and regulating the biological action of the polypeptide of the present invention.
  • Example 22
  • An 8-week-old female BALB / c mouse was immunized with the recombinant polypeptide derived from human hAgK114-lbb obtained by the method of Example 7-3, and Spleens were excised from the immunized mice to obtain antibody-producing cells.
  • antibody-producing cells and mouse myeloma-derived SP2 / 0-Ag14 cells were suspended in a serum-free medium, and both cells were mixed well. The washed cells were fused by a conventional method, and the hybridomas were selectively cultured. As a result, a culture supernatant showing immunoreactivity with the polypeptide of the present invention was confirmed.
  • Hybridomas were collected and cloned by applying the limiting dilution method.
  • the established hybridoma was cultured and analyzed according to a conventional method.
  • the monoclonal antibody produced a monoclonal antibody against the polypeptide of the present invention.
  • the monoclonal antibody of this example is used for the purification of the polypeptide of the present invention or the qualitative or quantitative detection of the polypeptide of the present invention, for example, a fluorescent immunoassay, an enzyme immunoassay. It is extremely useful in measurement methods. Further, it is useful in suppressing and regulating the biological action of the polypeptide of the present invention.
  • Example 2 3
  • Example 9 The same procedures as in Example 21 were carried out except that the mouse-derived recombinant polypeptide pAgAgK114--1FL obtained by the method of Example 13 was used as the antigen to be used for immunization. By operation, an antiserum containing a monoclonal antibody against the recombinant polypeptide was obtained.
  • the polyclonal antibody of this example is used for purification of the polypeptide of the present invention or qualitatively or quantitatively detecting the polypeptide of the present invention, for example, a fluorescent immunoassay, an enzyme immunoassay, etc. It is extremely useful in It is also useful for inhibiting the biological action of the polypeptide of the present invention.
  • Example 2 4 The polyclonal antibody of this example is used for purification of the polypeptide of the present invention or qualitatively or quantitatively detecting the polypeptide of the present invention, for example, a fluorescent immunoassay, an enzyme immunoassay, etc. It is extremely useful in It is also useful for inhibiting the
  • Example 9 was repeated except that a rat was immunized using the mouse-derived recombinant polypeptide smAgKI14-1 FL obtained by the method of Example 13 as an antigen to be used for immunization.
  • a clone of Hypri-Doma was established.
  • the established hybridoma was cultured and analyzed according to a conventional method, it produced a monoclonal antibody against the polypeptide of the present invention.
  • the monoclonal antibody of this example is extremely useful in the purification of the polypeptide of the present invention and the qualitative or quantitative detection of the polypeptide of the present invention, for example, in a fluorescent immunoassay, an enzyme immunoassay and the like. It is useful. Further, it is useful for suppressing and regulating the biological action of the polypeptide of the present invention.
  • Example 2 5 The monoclonal antibody of this example is extremely useful in the purification of the polypeptide of the present invention and the qualitative or quantitative detection of the polypeptide of the present invention, for example, in a fluorescent immunoassay, an enzyme immunoassay and the like. It is useful. Further, it is useful for suppressing and regulating the biological action of the polypeptide of the present invention.
  • CD-1 mice (8 weeks old, male) were anesthetized and the dorsal aorta was aseptically removed.
  • the obtained aorta was washed with PBS, and then cut into a ring having a width of about 1 mm with a sterilized scalpel.
  • the large artery fragment was allowed to stand still in a gelatin-coated 6-well plate, and 10% F
  • the cells were cultured in a D-MEM medium containing CS by a conventional method for 24 hours. After cultivation, the medium was removed, and a serum-free medium for endothelial cell growth containing 5 g / ml of a monoclonal antibody against mouse polypeptide obtained by the method of Example 24 was added, and cultivation was continued for another 10 days.
  • the antibody of the present invention may have an action of suppressing angiogenesis, and is useful for cancer caused by angiogenesis and diseases caused by cancer metastasis.
  • Sebocytes were prepared from the back skin of CD-1 mice (5 weeks old, male) by a conventional method.
  • the sebocytes, 6% FCS and 2% heat Bokuchi including Qing D- MEM: H am 's F 1 2 (1: 1) using a mixed medium was adjusted to 1 XI 0 3 pieces / cm 2, The seeds were seeded on a dish of 6 O mm in diameter.
  • a monoclonal antibody against the mouse polypeptide obtained by the method of Example 24 was added at a concentration of 1 Og / mI, and the cells were cultured for 14 days by a conventional method. The same treatment using a medium containing no antibody was used as a control.
  • a Nile Red staining solution adjusted to a concentration of about 100 n / mI with PBS was added to the collected sebum cells, and reacted at room temperature in the dark for 20 minutes. After completion of the reaction, the percentage of cells that formed fat globules (mainly composed of triglycerides, free fatty acids, cholesterol, and hexesters) in the cells was determined by flow cytometry analysis. Beta. As a result, it was found that the group to which the antibody was added had a lower rate of forming fat globules formed in sebocytes than the control group. Therefore, the antibody of the present invention suppresses the production of fat globules in sebocytes and is useful for alleviating dermatitis such as acne.
  • the antibody of the present invention in the form of a chimeric antibody is prepared as follows. First, a clone of a hybridoma producing a mouse monoclonal antibody against the human-derived polypeptide of the present invention obtained by the method of Example 22 was cultured according to a conventional method, and RNA produced by Biotechs was used. Total RNA derived from the hybridoma is prepared by a conventional method using a preparative reagent such as “Ultraspec LSII”, and the reverse transcriptase is reacted with the total RNA to obtain cDNA. Subsequently, PCR primers were designed with reference to the PCR primers described in S. 'Taran' The Jones et al., Biotechnology, Vol. 9, pp.
  • a cDNA fragment encoding the variable region in the light chain of the antibody and a cDNA fragment encoding the variable region in the heavy chain of the antibody are each amplified by PCR.
  • the amplified cDNA from each PGR product is recovered by polyethylen glycol precipitation or the like, and a plasmid such as “pCR — Script Cam SK (10)” is used. Clone to vector. Escherichia coli is transformed using the obtained vector, and the transformant is cultured to collect cells, and then the recombinant DNA is collected from the cells.
  • variable region in the light chain of the antibody is encoded by the usual didexy method.
  • c DN Decode the base sequence of cDNA encoding the variable region of A and the heavy chain of the antibody, and elucidate the amino acid sequence coding.
  • the light chain and heavy chain in the mouse antibody of the present invention are compared by comparing and matching the amino acid sequence thus identified with the amino acid sequence of the variable region already reported for the mouse antibody. The amino acid sequence of the variable region is determined.
  • DNA containing the nucleotide sequence encoding the constant region of the light chain of human immunoglobulin was transferred to Cell, Vol. 22 by Pierre Haiter et al. , Pp. 197 to 207 (1989), and isolated from a human gene library.
  • a DNA consisting essentially of the DNA encoding the constant region of the light chain is obtained by ordinary PCR.
  • human light constant region DNA a DNA encoding the variable region in the light chain of the mouse antibody cloned as described above (referred to as “mouse light chain variable region DNA”).
  • mouse light chain variable region DNA a DNA encoding the variable region in the light chain of the mouse antibody cloned as described above (referred to as “mouse light chain variable region DNA”).
  • human light chain constant region DNAs and mouse light chain variable region DNAs were designated as type III, and were reported by Robert 'Em' Photon et al. Applying the “overlap extension method” described in Vol. 17, pp. 270 to 279 (1993), downstream of the mouse light chain variable region DNA
  • the human light chain constant region DNA is ligated to obtain a DNA comprising a restriction enzyme recognition sequence at the 5 'end and the 3' end.
  • an origin of replication in Escherichia coli such as “pSV2-neo” (ATCC 37149), a motor that functions in mammalian cells, and / or an enhancer
  • a DNA containing a restriction enzyme recognition sequence, a selection sequence, and the like located under the control is prepared.
  • This expression vector and the DNA containing the human light chain constant region DNA and the mouse light chain variable region DNA obtained above were cut with restriction enzymes, mixed, and ligated using ligase. To obtain a recombinant DNA comprising DNA encoding the light chain of the chimeric antibody o
  • DNA encoding the constant region of the heavy chain (r-chain) of the human immunoglobulin belonging to the IgG class was collected by N. Tarikhaji et al., Cell, Vol. Volume, pages 671 to 679 (1992), and isolated from a human gene library.
  • the portion coding for the constant region of the heavy chain consists of four independent exons, as described in the article.
  • the isolated DNA is referred to as type I, and the above-mentioned “overlap extension method” is applied to the DNA to connect four exons (referred to as “human heavy chain constant region DNA”). ).
  • mouse heavy chain variable region DNA a DNA encoding a variable region in the heavy chain of the mouse antibody cloned as described above (referred to as “mouse heavy chain variable region DNA”) is obtained.
  • the human heavy chain constant region DNA and mouse heavy chain variable region DNA are referred to as type III, and the above-mentioned “overlap extension method” is applied to form the mouse heavy chain variable region DNA.
  • the human heavy chain constant region DNA is ligated downstream to obtain a DNA comprising a restriction enzyme recognition sequence at the 5 'end and the 3' end.
  • the replication origin in Escherichia coli such as “Jul 5 2 — 9 1:” (Hatch 0 0 3 7 1 4 5), functions in mammalian cells.
  • This expression vector and the human heavy chain constant Region DNA and DNA containing the mouse heavy chain variable region DNA are each cleaved with restriction enzymes, mixed, and ligated using ligase to comprise DNA encoding the chimera antibody heavy chain Obtain recombinant DNA.
  • the recombinant DNAs comprising the DNAs encoding the light and heavy chimeric antibodies described above were transferred to mammalian cell lines such as GHO-K1 cells (ATCCCCL-61). It will be introduced at the same time by the polling method.
  • a cell group obtained as a result of the DNA introduction is selected based on the selected sequence in the expression vector, and the selected cells are cultured. For each culture supernatant, the presence or absence of the ability to neutralize the biological action of the human-derived polypeptide of the present invention is examined by the method described in Example 11 or Example 18.
  • the limiting dilution method is applied to cells derived from the culture supernatant in which the desired neutralizing ability has been observed, and a single cell is obtained to obtain a transformant producing the chimeric antibody in the form of the antibody of the present invention.
  • the transformant is cultured while the culture scale is enlarged, and the antibody is purified from the culture supernatant according to a conventional antibody purification method to obtain the antibody of the present invention in the form of a chimeric antibody.
  • the thus obtained antibody of the present invention effectively exerts the ability to neutralize the human-derived polypeptide of the present invention, similarly to the mouse antibody against the polypeptide of the present invention.
  • the database was searched using a database for the framework structure of a human-derived antibody having homology to the framework structure of the chimeric antibody, and an amino acid similar to the human-derived framework structure having homology was confirmed. If the DNA in this example is modified and expressed so as to have an acid sequence, an antibody as a humanized antibody having a framework structure derived from human can be obtained. Further, based on the amino acid sequence of the humanized antibody thus obtained, a conventional software for protein structure analysis is used. The three-dimensional structure is predicted using the tween, and compared with the three-dimensional structure predicted in the same manner from the amino acid sequence of the original monoclonal antibody, a three-dimensional structure closer to the original mouse antibody is obtained.
  • a humanized antibody having substantially the same function as the original mouse monoclonal antibody can be obtained.
  • the chimeric antibody obtained according to the present example and the humanized antibody obtained by modifying such an antibody are useful for treating susceptible diseases.
  • This product enhances the expression of gelatinase in cells when applied to areas of skin damage such as wounds and inflammation, and improves various skin disorders such as skin wound treatment, topical dermatitis and contact dermatitis. It is useful as an external preparation for medicine.
  • the proliferation of hematopoietic cells is remarkably promoted, so that it is useful for the expansion of hematopoietic cells during bone marrow transplantation and the like.
  • Example 2 9. External skin cream>
  • Monostearic acid Polyethylene glycolyl serine 20 parts by weight Self-emulsifying glycerin monostearate 50 parts by weight Eicosanil behenate 10 parts by weight Liquid paraffin 19 parts by weight Trioctanoic acid trime 100 parts by mass of tilol propane
  • the following components except for the liquid composition were added and mixed to the above mixture according to the following composition, and the mixture was cooled to 30 ° C or lower, and then obtained in Example 28.
  • the resulting liquid composition was added in the following composition, and emulsified by a homogenizer to produce a skin external cream.
  • Example 3 1 It was prepared by the method of Example 22 in a physiological saline solution containing 1% (w / V) of high-purity hydrated crystal trehalose (trademark of “Treha”, trade name, sold by Hayashibara Corporation) as a stabilizer. Dissolve the antibody to a concentration of 1 mg / mI, remove pyrogen by a conventional method, and microfiltrate. The solution was obtained by sterilization.
  • the product having excellent stability is useful as an inhibitor of overexpression of gelatinase in skin cells and hyperproliferation of hematopoietic cells by the polypeptide of the present invention.
  • Example 3 1 Example 3 1
  • the polypeptide of the present invention has an activity of enhancing the expression of gelatinase in mammalian skin cells, and is used for treating skin wounds, or for dermatitis, atopic dermatitis, and contact dermatitis due to ultraviolet rays. It is extremely useful for improving various skin disorders, and as a medicine for regenerative medicine. Also, when used as cosmetics, it is effective in improving the therapeutic effect on skin diseases.
  • the polypeptide of the present invention has a remarkable action of promoting the proliferation of hematopoietic cells. It can be used as a hematopoietic cell proliferation promoter when expanding hematopoietic cells outside.
  • antibodies against the polypeptide of the present invention The present invention is extremely useful for purification of the polypeptide of the present invention and qualitative or quantitative detection of the polypeptide of the present invention, for example, in a fluorescence immunoassay, an enzyme immunoassay and the like. Further, the antibody of the present invention can be used for the purpose of suppressing the overexpression of gelatinase in skin cells and the excessive proliferation of hematopoietic cells. Furthermore, the antibody of the present invention can also be used as a therapeutic / prophylactic agent for susceptible diseases. The present invention is an invention having such remarkable effects, and is an invention having a great significance in contributing to the art.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Dermatology (AREA)
  • Hematology (AREA)
  • Diabetes (AREA)
  • Biophysics (AREA)
  • Obesity (AREA)
  • Biochemistry (AREA)
  • Birds (AREA)
  • Epidemiology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Cosmetics (AREA)

Abstract

L'invention concerne un moyen pour réguler efficacement l'expression de la gélatinase dans les cellules mammaliennes de la peau et/ou la prolifération des cellules hématopoïétiques. Cet objectif est réalisé par l'utilisation d'un polypeptide comprenant une séquence partielle d'acides aminés représentée par l'une des séquences SEQ ID NOS:1 à 3 dans le listage de séquences ou un polypeptide possédant une séquence d'acides aminés dérivée d'une séquence partielle d'acides aminés représentée par n'importe laquelle des séquences SEQ ID NOS:1 à 3 dans le listage de séquences par la délétion, la substitution et/ou l'ajout d'un ou de plusieurs acides aminés à l'intérieur d'une série choisie pour ne pas perdre l'effet de potentiation biologique de l'expression de gélatinase dans les cellules mammaliennes de la peau ou pour favoriser la prolifération de cellules hématopoïétiques, son anticorps et son utilisation.
PCT/JP2003/014161 2002-11-06 2003-11-06 Polypeptide physiologiquement actif et son anticorps ainsi que son utilisation WO2004042056A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP2004549627A JPWO2004042056A1 (ja) 2002-11-06 2003-11-06 生理活性ポリペプチドとその抗体及びそれらの用途
AU2003277583A AU2003277583A1 (en) 2002-11-06 2003-11-06 Physiologically active polypeptide and its antibody and use thereof

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2002322925 2002-11-06
JP2002-322925 2002-11-06
JP2003346463 2003-10-06
JP2003-346463 2003-10-06

Publications (1)

Publication Number Publication Date
WO2004042056A1 true WO2004042056A1 (fr) 2004-05-21

Family

ID=32314060

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2003/014161 WO2004042056A1 (fr) 2002-11-06 2003-11-06 Polypeptide physiologiquement actif et son anticorps ainsi que son utilisation

Country Status (4)

Country Link
JP (1) JPWO2004042056A1 (fr)
AU (1) AU2003277583A1 (fr)
TW (1) TW200418979A (fr)
WO (1) WO2004042056A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006137747A (ja) * 2004-10-14 2006-06-01 Hayashibara Biochem Lab Inc サイトカイン類及び／又はケモカイン類産生増強剤

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999055865A1 (fr) * 1998-04-29 1999-11-04 Genesis Research And Development Corporation Limited Polynucleotides isoles de cellules de la peau et leurs procedes d'utilisation
WO2000069884A2 (fr) * 1999-05-14 2000-11-23 Genesis Research & Development Corporation Limited Compositions isolees a partir de cellules cutanees, et leurs procedes d'utilisation
WO2001090357A1 (fr) * 2000-05-24 2001-11-29 Genesis Research & Development Corporation Limited Compositions isolees des cellules cutanees et methodes d'utilisation de ces compositions
WO2003016506A2 (fr) * 2001-08-17 2003-02-27 Incyte Genomics, Inc. Proteines secretees

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999055865A1 (fr) * 1998-04-29 1999-11-04 Genesis Research And Development Corporation Limited Polynucleotides isoles de cellules de la peau et leurs procedes d'utilisation
WO2000069884A2 (fr) * 1999-05-14 2000-11-23 Genesis Research & Development Corporation Limited Compositions isolees a partir de cellules cutanees, et leurs procedes d'utilisation
WO2001090357A1 (fr) * 2000-05-24 2001-11-29 Genesis Research & Development Corporation Limited Compositions isolees des cellules cutanees et methodes d'utilisation de ces compositions
WO2003016506A2 (fr) * 2001-08-17 2003-02-27 Incyte Genomics, Inc. Proteines secretees

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
AKIKO OKADA, ET AL: "EXPRESSION OF MATRIX METALLOPROTEINASE DURING RAT SKIN WOUND HEALING EVIDENCE THAT MEMBRANE TYPE-1 MATRIX METALLOPROTEINASE IS A STROMAL ACTIVATOR OF PRO-GELATINASE A", J. CELL. BIOL., vol. 13, no. 1, 1997, pages 67 - 77, XP002974864 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006137747A (ja) * 2004-10-14 2006-06-01 Hayashibara Biochem Lab Inc サイトカイン類及び／又はケモカイン類産生増強剤

Also Published As

Publication number Publication date
TW200418979A (en) 2004-10-01
AU2003277583A1 (en) 2004-06-07
JPWO2004042056A1 (ja) 2006-03-09

Similar Documents

Publication Publication Date Title
KR100903984B1 (ko) Aimp1 폴리펩티드 또는 이의 단편을 유효성분으로 포함하는 피부 노화 방지, 주름 개선, 피부 유연 증진 및 탄력 증진용 화장료 조성물
US7335641B2 (en) Method for stimulating hair follicle cell proliferation
JP7000161B2 (ja) 新規ペプチド及びこれを含む組成物
JP2002518347A (ja) 前立腺癌治療のための免疫療法組成物および方法
EP3046934B1 (fr) Protéine de fusion
EP2995626B1 (fr) Protéines de fusion recombinantes bifonctionnelles pour l'inhibition de l'angiogenèse dans le microenvironnement tumoral pour l'activation du système immunitaire adaptatif, gène et utilisations associées
JP2001505407A (ja) 腫瘍壊死因子関連リガンド
JP2012513775A (ja) ＤＬＫ１−Ｆｃ融合タンパク質を有効成分として含む癌転移抑制用組成物
EP3808762A1 (fr) Protéine de fusion liée à un peptide perméable aux cellules, et composition comprenant une protéine de fusion ou un peptide perméable aux cellules et un facteur de croissance de cellules épithéliales comme principes actifs
JP2022023216A (ja) ビメンチン由来のペプチドに特異的に結合する物質を含む皮膚疾患の予防及び治療用組成物
CN108948201B (zh) 抗人csf-1r单克隆抗体及应用
KR101948238B1 (ko) 미녹시딜과 펩타이드의 결합체
ES2262231T3 (es) Polipeptido expresado en la capa cornea de la epidermis y su utilizacion.
CN109096397B (zh) 一种抗人csf-1r单克隆抗体及应用
CN109053887B (zh) 一种抗人csf-1r单克隆抗体及用途
WO2004042056A1 (fr) Polypeptide physiologiquement actif et son anticorps ainsi que son utilisation
AU2004238713A1 (en) Oligopeptide
JP2005506383A (ja) ヒトの副甲状腺ホルモンに由来するペプチドにＴａｔペプチドが結合された融合ペプチド、その製造方法およびこれを含む経皮性体脂肪減量化粧品組成物
JPH10147515A (ja) 皮膚外用剤
CN113499434B (zh) 一种治疗发少、脱发的洗发水
KR102221365B1 (ko) 트롤록스-펩타이드 결합체 및 그의 용도
ITMI950676A1 (it) Vaccini polinucleotidici
KR102236127B1 (ko) 티로시나아제를 억제하는 항-티로시나아제 항체 및 이의 용도
JP2001017188A (ja) 新規なｖｅｇｆ／ｐｄｇｆ様因子
CN109053886A (zh) 抗人csf-1r单克隆抗体及用途

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP KR NZ US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2004549627

Country of ref document: JP

122 Ep: pct application non-entry in european phase