WO2004042056A1 - Physiologically active polypeptide and its antibody and use thereof - Google Patents

Abstract

It is intended to provide a means of effectively controlling the expression of gelatinase in mammalian skin cells and/or the proliferation of hematopoietic cells. The above problem is solved by providing a polypeptide having a partial amino acid sequence represented by any of SEQ IS NOS:1 to 3 in Sequence Listing or a polypeptide having an amino acid sequence derived from a partial amino acid sequence represented by any of SEQ IS NOS:1 to 3 in Sequence Listing by deletion, substitution and/or addition of one or more amino acids within such a scope as not substantially losing a biological effect of potentiating the expression of gelatinase in mammalian skin cells or promoting the proliferation of hematopoietic cells, its antibody and use thereof.

Description

 Description Bioactive polypeptides and their antibodies and their uses

 The present invention relates to a physiologically active polypeptide, its antibody, and its use. Background art

 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.

It is known that matrixmeta II oprotease (MMP) plays an extremely important role in cell development, differentiation, tissue formation and morphological changes, wound healing and tissue repair (eg, , Tatsuya Okamoto, "Activation Mechanism of Matrix Meta-oral Protease (MMP)", Biochemistry, 1991, Vol. 71, Vol. 12, No. 13 See). MMP is a generic term for zinc-requiring protease that hydrolyzes extracellular matrix constituent proteins in a neutral pH environment. It is a connective tissue cell (fibroblast), epithelial cell, inflammatory cell, and metastatic cancer Produced by cells, etc., more than 26 enzymes have been reported to date. 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.

 On the other hand, in recent years, 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. As is well known, 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. However, 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.

In order to solve this problem in bone marrow transplantation, healthy bone marrow cells are once removed from a living body and expanded The method of transplanting to a human is being studied earnestly. If this method is put to practical use, not only will it be easier to secure bone marrow, but also the amount of bone marrow extracted from the bone marrow donor can be significantly reduced, thereby reducing the mental and physical burden on the bone marrow donor, It is expected that the safety of bone marrow donation itself can be significantly improved.

 Currently, in the art, proliferation of hematopoietic cells using site force factors such as stem cell factor, FIk2 / FIt3 ligand, interleukin 6, trombopoetin, and intermouth 3 is being intensively attempted. However, since all of these cytodynamics have the property of inducing the differentiation of hematopoietic stem cells as well as the proliferation of hematopoietic cells, it has not yet been possible to obtain a desired amount of hematopoietic cells by this method. (For example, “New developments in research on hematopoietic stem cells '99-2000 stem cell development ■ New facts of differentiation and application to treatment”, Experimental Medicine (extra number), 1991, sheep See Earth Science Company, Vol. 7, No. 5, pp. 149 to 150). Disclosure of the invention

 In view of such circumstances, an object of the present invention is to provide a means for effectively enhancing or regulating the expression of gelatinase in mammalian skin cells.

 It is another object of the present invention to provide a means for effectively promoting or regulating the growth of mammalian hematopoietic 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.

 That is, 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.

 Further, the present invention solves the above-mentioned problem by providing a DNA encoding such a polypeptide.

 Further, 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.

 Further, the present invention solves the above-mentioned problem by providing an antibody against such a polypeptide.

Further, 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. In addition, 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. Furthermore, 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. BRIEF DESCRIPTION OF THE FIGURES

 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.

Explanation of reference numerals

hAgK114- 1 ac DNA: D encoding a polypeptide having the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing

N A

hAgK114- 1bc DNA: coding for a polypeptide having an amino acid sequence represented by SEQ ID NO: 5 in the sequence listing D

N A

FLAG: DNA encoding FLAG peptide

 S V 40 int r 0 n / p A: Simian virus 40 (S V

4 0) Splice poly A additional signal

 P o I y o m a 0 r i: Poliovirus DNA replication origin and enhancer

 SV40ori • SV40 DNA replication origin (excluding the origin)

 C o I E 1: colisin E 1

 M13ori-M13 phage DNA origin of replication

 SupF: Anno-sublesser gene

 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

p o I y A: poly A additional signal

 Amp: Ampicillin resistance gene

Ne0: neomycin resistance gene

 E F-1 promoter: elongation factor 1 alpha promoter dhfr: dehydrofolate reductase gene

 Pp0Iyh.edrin: Polyhedrin promoter The best mode for carrying out the 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 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. In the present invention, skin cells refer to epithelial cells, fibroblasts, mesenchymal cells and other skin cells, and 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. The 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. These 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. A polypeptide having a partial amino acid sequence represented by any one of SEQ ID NOs: 1 to 3 in the sequence listing, wherein one of the constituent amino acids is within a range that does not substantially lose gelatinase expression enhancing action or hematopoietic cell growth promoting action. It may be a mutant in which one or two or more amino acids are deleted, substituted with another amino acid, or further added with one or more amino acids. Examples of such 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. For example, 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. 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 Or 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.

 Among the polypeptides of the present invention, 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. As an example of the DNA 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. And DNA having a nucleotide sequence complementary thereto or a nucleotide sequence complementary thereto. Of course, DNA coding for the above-mentioned mutant of the polypeptide of the present invention 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. This is inserted into an appropriate cloning vector capable of autonomous replication, and the obtained recombinant cloning vector is introduced into an appropriate host such as Escherichia coli to obtain a transformant. The 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. At present, cDNA libraries derived from various cells are commercially available, and such commercially available libraries can also be used. On the other hand, in order to artificially synthesize 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. Can be Examples of such vectors include, for example, p K Κ 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. T i plasmid, R i plasmid, pBI121, pCDM8, pRc / CMV are preferred.

 In order to insert the DNA of the present invention into such a vector, 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. In order to ligate the DNA fragment and the vector fragment, if necessary, they 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. In order to obtain transformants, 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. On the other hand, when the host is an animal cell, 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. In order to select a target clone from the thus-formed transformants, 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. For the 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. And nitrogen-containing inorganic or organic substances such as oil, yeast, yeast extract, defatted soybean, corn steep liquor, and meat dex. Usually between 20 and 60, depending on the type of host cell and vector, 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.

 In order to produce the polypeptide of the present invention using a natural source, for example, 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. Certain PC-3 (ATCCCRL-1 4 3 5) cells, bile duct-derived cell lines HuCCTI (JCRB0425) cells, and lung cancer-derived cell lines CaIu-3 (ATCCHTB) — 55) Cells, KB (ATCCCCL-17) cells, which are cell lines derived from oral epidermoid carcinoma, are cells derived from Humus Yuichi, such as ovarian epithelial cells CH 0- K1 (ATCCCCL-61) cells, kidney-derived fibroblasts BHK-21 (ATCCCCL-10) cells, and 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. In addition, 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.

When a mammalian cell having the polypeptide-producing ability of the present invention is amplified using the in vivo cell proliferation method, 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.

 Further, the 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. As 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. 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 ^ ¾Jr? X electrophoresis. General methods are used, and these methods can be used if necessary. What is necessary is just to combine suitably. Then, depending on the final use form, the purified polypeptide may be concentrated and freeze-dried to a liquid or solid state.

 Further, 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. According to the present invention, 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. The term "immune reaction" as used herein 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. For example, 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. Usually, for example, rodents such as mice, rats, hamsters, egrets, and guinea pigs, mammals including artiodactyls such as goats and sheep, and 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. When immunizing rodents, 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”). Usually, during and / or after the 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.

 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. The term “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. . In order to obtain such eightybridoma, first, after immunization as described above, usually after about 3 to 5 days, 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. For the cell fusion, for example, a conventional method using a fusion promoter such as polyethylene glycol-Sendai virus or an electric pulse is appropriately employed. Next, 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. Using the culture supernatant of the propagated eightybridoma in accordance with a conventional method, immunity to the polypeptide of the present invention is tested.The presence or absence of reactivity is determined, and those showing the desired immunoreactivity can be selected. Thus, 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.

 As described above, the monoclonal antibody of the present invention also includes a so-called “humanized antibody” which is usually prepared by a protein engineering technique. To prepare 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. 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. In addition, 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. For example, 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. Usually, 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. Since 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. More specific cases where the effects of the polypeptide of the present invention can be expected are 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. In the cosmetics field, for example, it can be used for hair cosmetics, basic cosmetics, and the like.

In addition, 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. More specific cases where the effects of the polypeptide of the present invention can be expected 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. Since 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. In addition, when the polypeptide of the present invention is 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. On the other hand, when the polypeptide of the present invention is 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.

Although 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. 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.

Examples of the carbohydrate include sugars such as glucose, fructose, lactose, trehalose, maltose, sucrose, lactose, syrup, cyclic sugars such as cyclodextrin and cyclic tetrasaccharide, and erythri. One or two of sugar alcohols such as tall, mannitol, sorbitol, xylitol, maltitol, reduced starch syrup, natural polysaccharides such as pullulan, carrageenan, natural gums, and carboxymethyl cellulose By the addition of the above, not only solid ones can be advantageously used for shapeability but also advantageously for stabilization and taste improvement of the polypeptide of the present invention. In order to produce a composition comprising the polypeptide of the present invention, 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. As described above, 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. Although 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. In addition, in the gelatinase expression enhancement test, 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.

In addition, 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.

 When used as pharmaceuticals, for example, extracts, elixirs, capsules, granules, pills, eye ointments, buccal patches, suspensions, emulsions, plasters, suppositories, powders, Alcoholic drinks, tablets, syrups, injections, tinctures, eye drops, ear drops, nasal drops, lozenges, ointments, fragrances, nasal sprays, limonade, remnants Agents, liquid extracts, lotions, poultices, sprays, coatings, baths, patches, pastas, cataplasms and the like.

Further, the composition of the present invention can also be used as 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. In terms of 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. As 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.

 In order to produce a composition comprising the polypeptide of the present invention in the form described above, 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.

As described above, the composition containing the polypeptide of the present invention as an active ingredient exhibits a gelatinase expression-enhancing effect on mammalian skin cells. In this case, 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. Further, 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. On the other hand, 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. When 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. Although 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 μg 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. Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited by these examples. Example 1

Cloning of DNA encoding human-derived polypeptide and determination of nucleotide sequence>

 <Example 1-1: Cloning of DNA encoding human-derived polypeptide>

 Commercially available human placenta-derived cDNA library (HumanP

I acenta M arathon — Readyc DNA, manufactured by Nippon Vector Dickinson Co., Ltd.) and has the nucleotide sequence represented by SEQ ID NO: 18 or 19 in the sequence listing. Synthetic DNA and a synthetic DNA having the base sequence represented by SEQ ID NO: 20 in Sequence: ^ are used as PCR primers, and PGR Thermal Cycler (Applied Biosystems, Inc. Gene Am PPCRS ystem 9

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. As a result, in the primary PCR, a PCR primer having a base sequence represented by SEQ ID NO: 18 in the sequence listing was used, By PCR amplification using 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. Of these, the one with 0.5 kbp was named R27L and the one with 1.5 kbp was named R27H. On the other hand, in the primary PCR, a PCR primer having the nucleotide sequence of SEQ ID NO: 19 in the sequence listing was used, and in the 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 (ρΗ7.2), 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. Next, 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 錶, 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. As a result, 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. In addition, 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 ρρΒIuescript SK (-) j digested with restriction enzyme Ec0RV as above, and the nucleotide sequence was analyzed. As a result, R48 was a partial nucleotide sequence of cDNA composed of R23 and R27L. Also, R 5 0 was a partial nucleotide sequence of cDNA composed of R23 and R27H.

 <Example 1-2: Determination of base sequence and encoded amino acid sequence

>

 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: に お け る 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 nucleotide sequence represented by coded the amino acid sequence consisting of 176 residues, which was described in addition to the nucleotide sequence represented by SEQ ID NO: 12 in the sequence listing. In these amino acid sequences, the first to the 149th partial amino acid sequences were completely identical. Example 2

<Cloning and nucleotide sequence of DNA encoding hamster-derived polypeptide>

 <Example 2-1: Preparation of Antibody to Eight Muster Hair Follicle Constituent Cells

>

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. By the CELLEIA method, 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.

 <Example 2-2: Synthesis of Hamster Epidermal Cell c DNA>

The epidermal cells prepared from 4-day old eight Muster of dorsal skin, 3 7 ° C, under the conditions of 5% C 0 ₂ obtained by culturing day 6. 6 X 1 0 ⁷ pieces in Kera Chinosai Bok medium Total 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. Next, using mRNA 1 as type I, 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 Ω using Gene Pulser Unit (manufactured by Nippon Bild * 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.

 <Example 2-3: Cloning of DA encoding hamster-derived polypeptide>

A method of cloning a gene coding for a cell membrane protein using an antibody against the protein (B. Seed et al., Procedurals, The National Academy, Said, Sciences, Inc.) The United States, "Ability to Learn" (Proc. Natl. Acad. Sci. USA), 1987, 1984, 84, 3365-5, 3369 Screening was performed in accordance with). First, the library for burning, divided into 10 groups, was seeded on an L plate containing 50 Atg / mI of ampicillin, and cultured at 37 ° C. The obtained colony (5.1 × 10 ⁴ clones / group) was recovered using a 50 ml I-separation medium, and further cultured at 37 ° C. for 2 hours. After collection, the cells were transferred to QIAGENPI asmid Midi Kit (Qiagen Co., Ltd.). More plasmid DNA was prepared. The obtained DNA of group 0 is subjected to electroboration using Gene Pulsarunit (manufactured by Nippon Bio-Rad Laboratories, Inc.) under the conditions of 220 V and 960 F. Was introduced into COS-1 cells. Two to three days later, the adhered COS-1 cells were collected using a 5 mM MEDTA, 0.02% sodium azide solution to prepare a cell suspension. After adding the monoclonal antibody mAbK114-111 prepared in Example 211 at a concentration of SOtgZml to the cell suspension and reacting on ice for 1 hour, no binding was observed. 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.し た I left it. This was centrifuged at 15,000 rpm for 10 minutes, and the supernatant was recovered. After phenol treatment, 1 / ig yeast tRNA was added, and the DNA was recovered by ethanol precipitation. The recovered or NA was introduced into the colonies of the electrophoresis described in Example 2-2 to form colonies. This series of operations was defined as one baning cycle, and the ten groups of libraries were each subjected to three times of baning. 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.

 <Example 2-4: Determination of base sequence and encoded amino acid sequence>

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. 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. A database search was performed on the basis of the amino acid sequence described in SEQ ID NO: 13 in the sequence listing of ham Ag K114-1 to find that no polypeptide having the same amino acid sequence exists. In contrast, ham Ag KI 14 11 was a new substance. Example 3

 <Cloning of DNA encoding mouse-derived polypeptide and determination of nucleotide sequence>

<Example 3-1: Database search>

 Database search (BLAST analysis) based on the nucleotide sequence of cDNA encoding hamAgK111-111 obtained in Example 2 (SEQ ID NO: 13 in the sequence listing) As a result, four nucleotide sequences (accession numbers AK 055 58, BC 0 069 58, AK 0 0276) that are considered to encode mouse homologues were obtained. 7 and AK093336) were obtained. Sequence number in the sequence listing based on the base sequence upstream of the initiation codon of AK055558 (mouse adult female placenta cDNA) to clone cDNA encoding mouse homologue A forward-chain primer having the nucleotide sequence represented by 27 was synthesized.

 <Example 3-2: Synthesis of mouse epidermal cell c DNA>

The epidermal cells prepared from the ear of BALBZ c mice, 3 7 ° C, 5% C 0 obtained by culturing overnight in ₂ conditions 8. 8 X 1 0 ⁶ amino cultured epidermis keratinocytes Sai Bok medium Total 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. The resulting transformant was planted in a broth medium (PH 7.2), cultured with shaking at 37 ° C for 18 hours, and the transformant was collected from the culture. Plasmid DNA is prepared by the usual alkaline SDS method, and a clone containing the target PCR amplified fragment is selected. I chose.

 <Example 3-4: 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 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. Was. 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. In addition, 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. 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. Example 4

 <Cloning and nucleotide sequence of DNA encoding mouse-derived secretory polypeptide>

 <Example 4-11: Cloning of DNA coding for mouse-derived secretory polypeptide>

 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. Next, the resulting DNA fragment (PCR181) was digested with the restriction enzyme Ec0RV into a plasmid vector pT7BIue (manufactured by Merck Ltd.) to obtain a DNA ligation kit Ver.2. (Yukara Bio Inc.) to recombine in a conventional manner to obtain a recombinant DNA (designated as pTB-mAgK114-CR188). Using the obtained recombinant plasmid, an Escherichia coli recombinant cell (JM109, manufactured by Yubari Kalabai Sai Co., Ltd.) was transformed. The obtained transformant was inoculated into a heaploss medium (PH 7.2), cultured at 37 ° C for 18 hours with shaking, and then the transformant was collected from the culture and subjected to ordinary alkaline-SDS. Plasmid DNA was prepared by the method described above, and a clone having the desired cDNA was selected.

<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. An 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%. In addition, the 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. A database search was performed on the basis of the amino acid sequence described in SEQ ID NO: 17 in the sequence listing of the present polypeptide, and the cDNA disclosed by accession number AK002 677 was not It completely matched the amino acid sequence of the polypeptide whose function was unknown. Example 5

<Production of human-derived polypeptide> Example 5 — 1: Construction of Expression Vector>

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. As a result of confirming the nucleotide sequence by a conventional method, a 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.

 <Example 5-2: Transformation and culture of C0S-〗 cells>

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. First, 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. Next, 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.

<Example 5-3: Purification of polypeptide>

Using 100 ml of the culture solution of the transformed C0S-1 cells as a purification material, 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 ηm as an index to obtain about 0.15 mg of the polypeptide in a yield of about 80%. This polypeptide was named hAgK114-4aFL.

 Using the purified purified recombinant polypeptide, SDS-PAGE was carried out in the presence of a reducing agent according to a conventional method. As a result, a single polypeptide was found at a position corresponding to a molecular weight of 300 kDa to 100 kDa. Peptide bands were detected. The reason for the wide molecular weight range was thought to be the effect of sugar chain modification. Example 6

<Production of human-derived polypeptide>

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. — Script C am SK (+) is digested with restriction enzymes Bam HI and Not I to prepare Bam HI-Not I fragments, which are then transferred to a Baculovirus transfer vector (p). VL1393 'was ligated to the BamHI-NotI site downstream of the polyhedrin' promoter. The obtained recombinant vector was designated as "pVL13393-hAgK114-1aFL". FIG. 2 shows the basic structure of the constructed expression vector pVL1393-hAgK114-4aFL. Next, 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. 0.5 ml of 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. To further increase the titre of the virus, 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.

 <Example 6-2: Transfection and culture of insect cells>

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.)) Ί X 1 0 ⁸ pieces / Ί 0 m I Then, 200 nI of the recombinant virus solution obtained in Example 6-1 was added, and 1

Infection was performed for 1 hour with stirring every 0 minutes. Next, 30 ml of Express Five serum-free medium was added, followed by culturing at 27 ° C for 3 days to obtain a transfection solution.

 <Example 6-3: Purification of polypeptide>

Using the transfusion solution (740 ml) obtained in Example 6-2 as a purification raw material, 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. After sufficient 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 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. Was.

SDS-PAGE was performed using the obtained purified purified recombinant polypeptide 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 20 to 35 kDa was obtained. Band was detected. Examination of the N-terminal amino acid sequence of this purified polypeptide by 5 residues by a conventional method revealed that the polypeptide had the amino acid sequence represented by SEQ ID NO: 39 in the sequence listing. Was completely consistent with the 26th to 30th amino acid sequences of the amino acid sequence described in the sequence listing under SEQ ID NO: 11. From the above, out of the amino acid sequences described in SEQ ID NO: 11 in the sequence listing, the 1st to 25th amino acid sequences are signal sequences for secretion, and the 26th and subsequent amino acid sequences. The amino acid sequence of SEQ ID NO: 4, ie, the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing, was confirmed to be the amino acid sequence of the mature polypeptide. Example 7

 <Production of human-derived polypeptide>

<Example 7-1: Construction of expression vector>

 Using a primer having the nucleotide sequence shown in SEQ ID NO: 33 in the sequence listing as a primer for the complementary strand, 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. Using this expression vector, 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.

Example 7 — 2: Transformation and culture of C 0 S—〗 cells>

 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.

<Example 7-3: Purification of polypeptide>

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. The 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. The 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

 <Production of hamster-derived C-terminal FLAG addition secretion mutant polypeptide>

 <Example 8-1: Construction of expression vector>

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. Although the 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 (+)

(Stratagene, Inc.) and cloned into the SrfI site. The nucleotide sequence was confirmed by a conventional method. Was added. Since this DNA coded the amino acid sequence described in addition to the nucleotide sequence shown in SEQ ID NO: 14 in the sequence listing, it was originally intended to be 1st to 2nd 2nd of hamAgK〗 14-1 as intended. It was confirmed that a cDNA encoding a recombinant mutant fusion polypeptide (named as hamAgKI141-1d2FL) in which the amino acid sequence up to F and the AG sequence were fused was prepared. . 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 1α 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 μ 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. And form transformants CHO- K 1 cells 5% FCS - was suspended in H am 's F 1 2 medium, after 48 hours incubation under the conditions of 3 7 ° C, 5% C 0 2, medium Change to 5% FCS_Ham's F12 medium containing 400 g / mI of G—418 (trade name “dienecin”, manufactured by Invitrogen Corporation) for 2 days. The culture was performed for 14 days while changing the medium once. EIA of culture supernatant using anti-FLAGM2 antibody labeled with horseradish peroxidase was used to transform CHO—K1 cells from CHO—K1 cells that had been transformed. One cell line was selected, single cloned by a conventional method, and named CΗ0—hamAgΚ114-1d2FL. 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.

 <Example 8-3: Purification of polypeptide>

 Purification of FLAG-added mutant polypeptide hamAgK1 14 — 1d2FL using CHO-hamAgK1114-1d2FL cell culture broth 20 I as a raw material for purification was as follows. I went to. The culture supernatant obtained by centrifuging the culture solution was concentrated 100-fold with an ultrafiltration membrane (trade name “Microza”, manufactured by Asahi Kasei Corporation), and then 0.1M sodium chloride was added. The antibody was applied to an anti-FLAGM2 antibody agarose column (volume: 5 ml, Sigma-Aldrich) pre-equilibrated with 50 mM Tris-HCl buffer (pH 7.4) containing lithium, and the FLAG sequence was added. 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.

SDS-PAGE was performed using the obtained purified purified recombinant polypeptide in the presence of a reducing agent according to a conventional method. As a result, a single polypeptide was located at a position corresponding to a molecular weight of 50 to 100 kDa. Peptide band Was detected. The reason for the wide molecular weight range was thought to be the effect of sugar chain modification. Examination of the N-terminal amino acid sequence of this purified polypeptide by 5 residues by a conventional method revealed that the polypeptide had the amino acid sequence represented by SEQ ID NO: 40 in the sequence listing. Was completely consistent with the 27th to 31st amino acid sequences of the amino acid sequence described in the Sequence Listing along with the nucleotide sequence represented by SEQ ID NO: 14. From this, out of the amino acid sequences described in the sequence listing as SEQ ID NO: 14, the first to 26th amino acid sequences are signal sequences for secretion, and 22 It was confirmed that the second amino acid sequence, that is, the amino acid sequence represented by SEQ ID NO: 7 in the sequence listing, was the amino acid sequence of the mature polypeptide. The polypeptides having the amino acid sequences of SEQ ID NOs: 6 and 7 in the sequence listing have the partial amino acid sequence of SEQ ID NO: 2 in the sequence listing in common. Was determined to be important. Example 9

 <Production of mouse-derived C-terminal FLAG addition-secretion mutant polypeptide>

 <Example 9-1: Construction of expression vector>

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. In order to obtain a secreted polypeptide, 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. In the amino acid sequence described along with the nucleotide sequence shown, 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. We decided to construct an expression vector in which the cDNA was modified so as to obtain a fusion polypeptide.

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.). As a result of confirming the nucleotide sequence by a conventional method, 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. It was confirmed that cDNA encoding a recombinant secretory mutant fusion polypeptide having a FLAG sequence at the C-terminus of the amino acid sequence (designated smAgK114-1FL) was prepared. The Xh0I—NotI fragment containing the cDNA was excised again, and the expression vector p REF—XN downstream of the EF-1α 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.

 <Example 9-2: Transformation and culture of CHO—K1 cells>

 Example 9 Expression Vector pRER—smAgKI1 Obtained in 11

4 FL is converted to CHO by the election port method as follows.

— Transformed into K 1 cells. First, Plasmid D

And NA 1 0 At g, premixed conventional method CHO- K 1 cells which had been prepared by the the liquid 8 0 0 ^ I containing 1 x 1 0 ⁶ cells in elect port Poreshi Yo emissions for cuvette in Bok It was left at ice temperature for 10 minutes. Next, the electoral port po- ration was performed by applying an electric pulse of 25 t FD and 100 V twice twice at one-minute intervals. Suspend the CH 0 one K 1 cells transformants conversion to 5% FCS- H am 's F 1 2 medium, after 48 hours incubation under the conditions of 3 7 ° C, 5% CO 2, the medium 4 The medium was replaced with a 5% FCS-Hams F12 medium containing 0 g / mI of G—418, and the cells were cultured for 8 days while changing the medium once every two days. EIA of culture supernatant using anti-FLAGM2 antibody labeled with horseradish peroxidase was used to transform transformed CHO-K1 cells that express high sm Ag KI14-1 FL from CHO-Κ-Ί cells. 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. Purification of the peptide smAgKI14--1FL was performed as follows: The culture supernatant obtained by centrifuging the culture solution was filtered through an ultrafiltration membrane (trade name "Microcloser", Asahi Kasei Corporation) ), And then equilibrated with a 50-m Tris-HCl buffer containing 0.15 M sodium chloride (H7.4). The mixture was applied to an agarose column (volume: 5 ml, manufactured by Sigma-Aldrich Corp.) to specifically adsorb the recombinant fusion polypeptide having the AG sequence, and then sufficiently washed with the same buffer to remove non-adsorbed components. 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.

Using the purified purified recombinant polypeptide, SDS-PAGE was carried out in the presence of a reducing agent according to a conventional method. A single polypeptide was obtained at a position corresponding to a molecular weight of 500 to 100 kDa. Band was detected. The reason for the wide molecular weight range was thought to be the effect of sugar chain modification. The N-terminal amino acid of this purified polypeptide is When the amino acid sequence was examined for 5 residues, it was found that the amino acid sequence had the amino acid sequence represented by SEQ ID NO: 41 in the sequence listing. It was completely identical to the 27th to 31st amino acid sequences of the amino acid sequence described in the sequence. From this, of the amino acid sequence described along with the base sequence represented by SEQ ID NO: 16 in the sequence listing,

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

 <Production of mouse-derived secretory polypeptide>

Example 10 0-1: Construction of Expression Vector>

 Recombinant plasmid pTB—mAgKI14PCR18 110〗 ng obtained in Example 4 was designated as 錶, 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.). As a result of confirming the nucleotide sequence by a conventional method, 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.

Named 4b. The constructed expression vector pRER — mAgK1

Fig. 6 shows the basic structure of 14b.

 <Example 10-12: Transformation and culture of CH0-K1 cells>

 Example 10 Expression vector obtained in 0-1 pRER-mAgK11

Using 4b, transformant CH 0—K 1 cells and mouse-derived peptide smAgK114–1 EIA using rat antiserum against FL mAgK1Ί4—1 In the same manner as in Example 9 except that a transformed CHO-K1 cell that highly expresses b was selected, a transformed CH0-K1 cell was obtained to obtain CHO—mAgΚ1 14 b1

6 1 3 8 The cells were plated on 5% FCS-RPMI 1640 medium containing 34.5 mg / L L-proline,

They were cultured in ^{3 7 0 C, 5% C} o 2 presence. After the cells became confluent β, the medium was changed to ASF medium and cultured for 3 days.

<Example 10-13: Purification of Polypeptide>

C Η m -mAg K11 4 b16-38 Purification of mouse-derived polypeptide mAgK114-1b using culture medium 201 of 8 cells as the raw material for i I went like that. The culture supernatant obtained by centrifuging the culture solution was subjected to a WG with a volume of 360 mI which had been equilibrated with phosphate buffered saline (PBS) (pH 7.5). Sepharose (Manufactured by Amersham Biosciences) The polypeptide was specifically adsorbed on the column. After thoroughly washing with the same buffer to remove non-adsorbed components, 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). When the polypeptide was recovered using the absorbance at 280 nm as an index, about 0.5 mg of the polypeptide was obtained. Using the purified recombinant polypeptide obtained, SDS-PAGE was carried out in the presence of a reducing agent according to a conventional method. A single polypeptide was found at a position corresponding to a molecular weight of 35 to 70 kDa. Bands were detected. By examining the N-terminal amino acid sequence of this purified polypeptide at 5 residues by a conventional method, it was found that it had the amino acid sequence represented by SEQ ID NO: 41 in the sequence listing. Was completely identical to the 27th to 31st amino acid sequences of the amino acid sequence described in the sequence listing together with the nucleotide sequence represented by SEQ ID NO: 17. From the above, of the amino acid sequences described together with the nucleotide sequence of SEQ ID NO: 17 in the sequence listing, the amino acid sequences from the 1st to the 26th are signal sequences for secretion, and the 27th and the following The amino acid sequence of SEQ ID NO: 10, ie, the amino acid sequence represented by SEQ ID NO: 10 in the sequence listing, was confirmed to be the amino acid sequence of the mature polypeptide. Polypeptides having the amino acid sequences represented by SEQ ID NOs: 8 to 10 in the sequence listing Have the partial amino acid sequence represented by SEQ ID NO: 3 in the sequence listing in common, and this amino acid sequence was determined to be important for biological action. Example 11

Of human-derived polypeptide enhances gelatinase expression in human-derived fibroblasts>

 Using the purified human-derived recombinant polypeptide hAgK114-1aFL obtained by the method of Example 5-3, the effect of enhancing the expression of gelatinase in human-derived fibroblasts was examined. According to a conventional method, fibroblasts (N) from normal human neonatal foreskin skin were placed in a 12-well microplate.

The HDF) at a concentration of 3 X 1 0 ⁴ pieces / ml 1 ml of M Edium

Seed into 106 medium (Cascade Bio Mouth Dix), 37 ⁰ C,

The cells were cultured for 2 days in the presence of 5% CO ₂ gas. Then the proliferated N

In HDF, adjust the purified hAgK111-111a FL purified sample obtained by the method of Example 5-3 to a final concentration of ΊtgZmI or 10fig / mI. 1 ml of the serum-free D-MEM medium was added, and the cells were further cultured at 37 ° C in the presence of 5% CO ₂ gas for 2 days. After completion of the culture, the culture supernatant was recovered by centrifugation and used for the measurement of gelatinase activity and gelatinase A protein. Culture supernatant obtained by culturing in the same manner except that FLAG peptide was added to a final concentration of 1 g / mI instead of the recombinant hAgK114-1-1a FL purified sample Was used as a control.

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 晚 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. The expression level of 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. (Daiichi Fine Chemical Co., Ltd., trade name: “anti-human MMP-2 antibody”), and further diluted 100-fold as a secondary antibody with anti-mouse immunoglobulin, western horseradish oxidase. The antibody was treated with an antibody (manufactured by Dako Japan KK), and lyseratinase A protein was detected using an ECL western blotting detection reagent (manufactured by Amersham Bay Saisense Co., Ltd.). The intensity of the band detected by the Western blotting method was measured all over the densitometer, and the expression level of gelatinase A protein in the control was evaluated as 100 relative to the control. Table 1 shows the results. table 1 Recombinant hAgK114-1aFL Gelatinase Gelatinase A

 Concentration (tgZm 1) Relative activity (%) Relative protein amount (%)

 0 * 100 100

 1 94 301

10 150 922

* Control: FLAG peptide was added to 1 gZml.

As is evident from the results in Table 1, 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. At a concentration of 0 tg ZmI, the expression of gelatinase A protein in NHDF was remarkably enhanced by 9 times or more in a dose-dependent manner. On the other hand, in the gelatinase activity evaluated by the activity staining method, the recombinant polypeptide hAgK114--1aFL was almost the same as the control at a concentration of 1 μg / mI and 10 μg / ml. The concentration of mI was about 1.5 times that of the control. In this study, recombinant polypeptide hAgK1141-1aFL significantly increased gelatinase A protein expression in NHDF in a dose-dependent manner, resulting in increased gelatinase activity. Has been found to enhance. Since the polypeptide of the present invention remarkably enhances the expression of gelatinase in human-derived fibroblasts, it can be used to promote the healing of wounds and inflammation in the skin. Example 1 2

 <Effect of polypeptide on gelatinase expression in mammalian fibroblasts and mesenchymal cells>

Humans, hamsters and mice obtained by the methods of Examples 5 to 0 Using purified purified recombinant polypeptides from humans, the effect of enhancing the expression of gelatinase in human and hamster-derived fibroblasts and mouse-derived mesenchymal cells was examined. According to a conventional method, fibroblasts (NHDF) 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. 9) narrowing seeded in medium containing growth factor I ml at a concentration of 3 X 1 0 ⁴ or Zm l, and cultured for 2 days at 3 7 ° C, 5% C 0 2 presence. Next, the purified polypeptide prepared by the method of Examples 5 to 10 was adjusted to the final concentration of 10 jg / mI, respectively, to the grown NHDF, FB or SC 9-19. 1 ml of D-MEM medium was added, and the cells were further cultured at 37 ° C in the presence of 5% CO ₂ for 2 days. After completion of the culture, the culture supernatant was recovered by centrifugation and used for measuring gelatinase activity. A culture supernatant obtained by culturing in the same manner as above was used as a control, except that the FLAG peptide was added to a final concentration of 10 g ZmI instead of the purified recombinant polypeptide.

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

 I: Not tested.

As is evident from the results in 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 hAgκ1114- 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

1 1 4-1 FL and mAg K 1 1 4-1b are all SC 9

-The gelatinase activity at 19 was increased about 1.3 times. In addition, both the human-derived polypeptide hAgΚ114-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. Example 13

Enhance Gelatinase Expression and Promote Wound Healing by a Mouse-Derived Polypeptide>

Anesthesia of 24 8-week-old male CD-1 mice and depilation of the back skin, followed by two 12-mm linear wounds before and after each midline, and left and right across the midline ^Two open wounds of 8 mm2 were made. Immediately after wound creation, 1 day, and 2 days later, one of the wounds was treated at one site with 8 g of the mouse-derived polypeptide smAgKI14-1 FL prepared by the method of Example 9-13. fx I dissolved in phosphoric acid buffer solution). For the remaining 16 mice, 8 mice were similarly coated with 0.2 Atg of FLAG peptide, and 8 mice were similarly coated with 4 g of human serum albumin, and used as controls. Seven days after wound creation, the size of each wound was measured, skin cells were collected from the wound site, and the cells were disrupted.The supernatant of the centrifuged supernatant was used to express gelatinase in the same manner as in Example 11. And evaluated by the active dyeing method. Table 3 shows the results. Table 3 Gelatinase expression enhancement

 BI. Materials Enhancement of gelatinase expression Gelatinase Number of individuals (8 animals) Relative activity (%) ゥ Serum albumin (control) 0 100 *

FLAG peptide 0 103 * derived from mouse smAgKI 14-1FL 5 170 **

 Wound area 7 days after wound sample

Linear wound (mm ² ) * Open wound (mm ² ) * ゥ Serum albumin (control) 29 32

 FLAG peptide 25 30 Mouse-derived smAgKI 14-1FL 14 20

 * 'Average of 8 animals

 **, average of 5 animals

As is clear from the results in Table 3, in the mouse-derived polypeptide smAgK111-FL-applied group, 5 out of 8 mice showed an effect of enhancing the expression of gelatinase, and the degree of the enhancement was about 1%. 7 times. In addition, 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 (群 serum albumin group). The values are shown. In addition, 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.

Example 14

<Enhancement of gelatinase expression by human-derived polypeptide and wound healing Acceleration>

Two 7-week-old male CD-II mice were anesthetized, the back skin was depilated, and two 15 mm linear wounds were made to the left of each midline. Immediately after wound creation, 1 day, and 2 days later, one of the two animals had one wound. The human-derived polypeptide hAgK114--1aFL prepared by the method of Example 5-3 was added to 1Q. β g (10 fi I of 10% glycerin dissolved in PBS) was applied thereto. For the remaining 10 mice, 10 tg / 10 ULI-10% glycerin PBS was added in the same manner as described above, and PBS was added and applied for control. Seven days after wound creation, the size of the wound was measured. In addition, the expression of gelatinase A in the wound site of the mouse was evaluated by the following immunostaining method. First, 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. Next, 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. Subsequently, the endogenous peroxidase was deactivated by treatment with methanol containing 0.3% hydrogen peroxide, and then a blocking agent (trade name “Block Ace ™”, Dainippon Pharmaceutical Co., Ltd.) ) At room temperature for 30 minutes. Next, 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. At room temperature for 1 hour. Wash with PBS After that, color development was carried out using a Persian oxidase color reagent (Siquid DAB—B lack Substrate Kit, manufactured by Zymed). The color development is stopped by washing with water, the nucleus is stained with hematoxylin (Wako Pure Chemical Industries, Ltd.) in the usual manner, and then sealed with a sealant (trade name "Enteran", Merck Ltd.). After processing, the stain was observed under a microscope. The results of immunostaining were visually observed for the degree of staining, and were evaluated as "one" (not stained), "ten" (slightly stained), "++" (slightly stained) and "++++" ( (Strongly stained). Table 4 shows the results. Table 4

 *,Average value

As is clear from the results in Table 4, the expression of gelatinase A protein in the wound was 9% in 10 out of 10 cases in the control group, and +10 '', 5 cases showed Γ + + '', 3 cases showed `` + '', whereas human-derived polypeptide hAgK1 14-1 & 1 in 12 cases In one animal, 6 cases showed +++, 4 cases showed Γ + 10, and 1 case showed `` 10, '' indicating that the application of human-derived polypeptide clearly enhanced the expression of gelatinase A protein. Was. Also, human Bok derived polypeptides h A g K 1 1 4 in the area of the linear wound -. 1 a FL applied group and 6 0 mm ² after 7 days the wound, 1 0 the control group (© shea serum albumin group) The value was significantly smaller than that of 5 mm ² . It was determined that the human-derived polypeptide hAgKl〗 4— —aFL enhanced the expression of gelatinase in mouse skin cells, and as a result, promoted the healing of skin wounds. Example 15

 <Hamster-derived polypeptide promotes proliferation of hamster bone marrow cells>

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 μm 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. Each solution was adjusted to a concentration of 5, 25 tg / ml and added to each solution at 100 I to give a total volume of 200 l (final concentrations of the polypeptides were 6.25 and 12.5 ng). / m I). After cultivation for 6 days, a dye that is an oxidation-reduction indicator (Trek's Diagnostics Co., Ltd.) Using the product name “Alama-I'm-Bul-I (aI amar BI ue)”), measure the fluorescence intensity using 544 nm as the excitation wavelength and 590 nm as the measurement wavelength, and use this fluorescence intensity to measure the cell proliferation. It was evaluated as an indicator. The control was performed without adding the polypeptide, and the relative fluorescence intensity when the polypeptide was used, with the fluorescence intensity of the control being 100%, was expressed as a relative cell growth rate as a percentage. Table 5 shows the results. Table 5

表 5の結果から明らかなように、 ポリペプチド濃度 6. 2 5 g /m l 及び 1 2. 5 g /m I のとき、 骨髄細胞の増殖は 対照を 1 0 0 %とした場合、 それぞれ約 2 2 5 %、 3 6 8 %で あり、 試験したハムスター由来ポリペプチド h a m A g K I 1 4 - 1 d 2 F Lは用量依存的にハムスター骨髄細胞の増殖を促 進した。 本ポリペプチドはハムスターをはじめとする哺乳類由 来の造血細胞の増殖を顕著に促進する生物作甩を有している。 実施例 1 6

As is clear from the results in Table 5, when the polypeptide concentrations were 6.25 g / ml and 12.5 g / ml, the proliferation of bone marrow cells was about 2% when the control was 100%. The tested hamster-derived polypeptide hamAgKI14-1d2FL promoted the proliferation of hamster bone marrow cells in a dose-dependent manner. This polypeptide has a biological effect that remarkably promotes the growth of hematopoietic cells derived from mammals such as hamsters. Example 16

 <Effect of mouse-derived polypeptide to promote mouse bone marrow cell proliferation>

Bone prepared from femur of BALB / c mouse (five weeks old, female) Medullary cells were prepared at a concentration of 7.6 × 10 ^s / ml using α-minimal medium (α Μ Ε 培 地 medium) containing 20 (V / V)% FCS, and coated with collagen. 100 I was seeded into a 4-well chamber. Next, the mouse-derived polypeptide smAgΚ1 14-1 FL obtained in Example 9 was similarly used in an αΜΜ medium containing 20 (v / v)% FCS for 10 Og ZmI. The resulting solution was adjusted to a concentration of 100 μl, and 100 I was added thereto to make a total volume of 200 μI (the final concentration of the polypeptide was 50 μg / mI). After culturing for 4 days, 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

表 6の結果から明らかなように、 ポリペプチド濃度 5 0 ^ _g /m I の条件下で、骨髄細胞の増殖は対照を 1 0 0 %とした時、 約 2 6 8 %であり、 試験したマウス由来ポリペプチド s m A g K 1 1 4 — 1 F Lはマウス骨髄細胞の増殖を顕著に促進した。 本ポリペプチドはマウスをはじめとする哺乳類由来の造血細胞 の増殖を顕著に促進する生物作用を有している。 実施例 1 7

As is evident from the results in Table 6, under the conditions of the polypeptide concentration of 50 ^ _g / mI, the proliferation of bone marrow cells was about 268% when the control was set at 100%, and the test was performed. The mouse-derived polypeptide smAgK114-FL significantly promoted the proliferation of mouse bone marrow cells. This polypeptide has a biological effect that remarkably promotes the growth of hematopoietic cells derived from mammals such as mice. Example 17

 <Promoting action of mouse bone marrow cell proliferation by a polypeptide derived from Humus Yuichi>

Bone marrow cells prepared from the femur of BALB mice (five weeks old, female) 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). After culturing for 6 days, the proliferated cells were fixed with 4% paraformaldehyde, treated with methanol according to a conventional method, and subjected to Giemsa staining. Next, 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. The control was 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%. The results are shown in Table 7. Table 7 Polypeptide Cell count Relative cell count

 Concentration (gZml) (pcs) (%)

 0 27 100

 12.5 100 370

As evident from the results in Table 7, under the conditions of the polypeptide concentration of 12.5 g ZmI, the proliferation of bone marrow cells was about 3700% when the control was set to 100% and tested. Hamster-derived polypeptide hamAgKI14—1d2FL also significantly promoted the proliferation of bone marrow cells from different mouse species. This polypeptide has a biological action that remarkably promotes the proliferation of hematopoietic cells derived from mammals such as mice and mice.

Example Ί 8

 <Promotion of mouse bone marrow cell proliferation by human-derived polypeptide

>

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. The solution was adjusted to a concentration of 5 g / mI, and 200 μl of this was added to obtain a total volume of 400 I (final concentration of the cells was 3.75 × 10 ⁵ cells / ml, Polybe The final concentration of the peptide is 2.5 / _6 g / ml). 3 7 ° C, 5% C 0 after 6 days of culture in ₂ the presence, expanded cells were fixed with 4% paraformaldehyde aldehydes, after methanol treatment by a conventional method, was subjected to formic厶Za staining. Next, this micrograph was taken, and 12 fields were randomly selected to count the number of cells, 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 as a control was set at 100%, and the relative cell number was expressed as a percentage. The results are shown in Table 8. Table 8

表 8の結果から明らかなように、 ポリペプチド濃度 2. 5 n g / m I の条件下で、 骨髄細胞の増殖は対照を 1 0.0 %とした 時、 約 1 4 4 %であり、 試験したヒ 卜由来ポリペプチド h A g K 1 1 4 ― 1 a F Lは種の異なるマウス由来の骨髄由来細胞の 増殖を顕著に促進した。 本ポリペプチドはヒ 卜、 マウスをはじ めとする哺乳類由来の造血細胞の増殖を顕著に促進する生物作 用を有している。 実施例 1 9

As is evident from the results in Table 8, under the conditions of the polypeptide concentration of 2.5 ng / ml, the proliferation of bone marrow cells was about 144% when the control was set to 10.0%. The mouse-derived polypeptide hAgK114-1aFL markedly promoted the proliferation of bone marrow-derived cells from different species of mice. This polypeptide has a biological effect that remarkably promotes the growth of hematopoietic cells derived from mammals such as humans and mice. Example 19

Acute toxicity test>

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

 <Mouse skin patch test>

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 μl 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. As a result, no abnormality was observed in the skin of any of the samples even at the maximum dose of 50 g Z patch tested. This result indicates that the polypeptide of the present invention is a substance that is safe even if used regularly on the skin of mammals including humans. Example 2 1

 <Preparation of polyclonal antibody>

 J W ゥ herons (female, weighing 2.5 k) were immunized according to the following schedule. As the first immunization, 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. Blood was collected from the ear vein of rabbits by a conventional method, and the immunoreactivity of the serum separated from the blood with the antigen was examined by a conventional method to confirm the expected increase in the antibody titer. Subsequently, the antigen was injected once intravenously at a dose of 100 g / animal without adjuvant. Seven days after the end of the final immunization, whole blood was collected from the carotid artery to obtain antiserum. The increase in the expected antibody titer in the serum of the egret by the above procedure indicates that this serum is an antiserum containing the polyclonal antibody against the polypeptide of the present invention. Therefore, according to the method of this example, a polyclonal antibody that reacts with the polypeptide of the present invention can be prepared.

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

Preparation of Monoclonal Antibodies>

 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. Next, antibody-producing cells and mouse myeloma-derived SP2 / 0-Ag14 cells (ATCCCRL1581) 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. As a result, 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

Preparation of Polyclonal Antibodies>

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

Preparation of Monoclonal Antibodies>

 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. In the same way as above, a clone of Hypri-Doma was established. When 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

 <Antibody proliferation inhibitory action of vascular endothelial cells>

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. Next, 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. did. A control treated similarly using a medium containing no antibody was used as a control. After cultivation, the proliferation of endothelial cells proliferating from the blood vessels was visually observed using a phase contrast microscope. As a result, in the test group to which the antibody was added, proliferation of endothelial cells was clearly suppressed as compared with the control. 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. Example 26

 <Sebum production inhibitory effect of antibody>

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. To this, 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. After the culture, 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. Example 2 7

 <Chimeric and humanized antibodies>

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. 88-89 (1991). Then, 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. Next, 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. The 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. Next, 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.

 Subsequently, 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. Next, using the isolated DNA as type I, a DNA consisting essentially of the DNA encoding the constant region of the light chain is obtained by ordinary PCR.

(Referred to as “human light constant region DNA”). Subsequently, 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”) is obtained. These 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. On the other hand, as an expression vector, 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

Separately, 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. In the isolated DNA, 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”). ). Subsequently, 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. On the other hand, as an expression vector, 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. Prepare a DNA containing a promoter and / or enhancer, a restriction enzyme recognition sequence located under the control thereof, a selection sequence, and the like. 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.

Next, 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. In addition, 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. If the DNA is further modified to have the expression, 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. Example 2 8

<Liquid composition>

 0.1% by mass of purified recombinant fusion polypeptide hAgK11-1a FL prepared in the same manner as in Example 5 and 0% by mass of human serum albumin in physiological saline. After dissolving to 1% by mass, the solution was sterilized by microfiltration according to a conventional method to obtain a liquid composition.

 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. In addition, when added to a medium during the culturing of hematopoietic cells, 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>

 The following ingredients are heated according to the following formula, using the usual method.

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.

 1, 3 — Butyrene glycol 50 parts by mass Sodium lactate 100 parts by mass Nori sekiseki rest 0 1 part by mass Peach leaf kiss 15 parts by mass Purified water 5 2 2 parts by mass Example 2 8 people 100 parts by mass This cream is

Latinase expression

Useful as an external skin cream to alleviate symptoms such as dermatitis

, Ma, there. Example 30

<Liquid>

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

 <Dry injection>

 100 mg of the humanized antibody obtained according to the method of Example 27 was dissolved in 100 ml of physiological saline containing 1% (w / V) of sucrose as a stabilizer, and the solution was dissolved in a conventional manner. After removing bacteria by microfiltration, dispense〗 mI into vials, freeze-dry, and stopper tightly. This product, which has excellent stability, is useful as a dry injection for treating and preventing susceptible diseases. This product is also useful as an angiogenesis inhibitor or sebum production inhibitor. Industrial applicability

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. In addition, 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. On the other hand, 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.

Claims

The scope of the claims 1. A polypeptide having a partial amino acid sequence represented by any one of SEQ ID NOS: 1 to 3 in the sequence listing. 2. The polypeptide according to claim 1, which has a biological effect of enhancing gelatinase expression in mammalian skin cells and / or a biological effect of promoting proliferation of mammalian hematopoietic cells.  3. In the partial amino acid sequence represented by any one of SEQ ID NOs: 1 to 3 in the sequence listing, the polypeptide has an amino acid sequence within a range that does not substantially lose the intended biological action. Claims wherein at least two or more have a deleted, substituted and Z or added amino acid sequence The polypeptide according to item 2.  4. The polypeptide according to any one of claims 1 to 3, which has an amino acid sequence represented by any one of SEQ ID NOS: 4 to 10 in the sequence listing.  5. A DNA encoding the polypeptide according to any one of claims (1) to (4).  6. The DNA according to claim 5, wherein the DNA has a base sequence represented by any one of SEQ ID NOS: 11 to 17 in the sequence listing, or a base sequence complementary to the base sequence.  7. One or more bases in the base sequence represented by any one of SEQ ID NOS: 11 to 17 in the sequence listing are other bases as long as the DNA does not change the amino acid sequence to be coded. 7. The DNA according to claim 6, wherein the DNA is replaced by: 8. The method according to claim 5, wherein the polypeptide is obtained by artificially expressing the DNA according to claims 5 to 7. 5. The polypeptide according to any one of paragraphs 1 to 4. 9. The step of culturing a cell or microorganism capable of producing the polypeptide according to any one of claims 1 to 4 and 8, and the step of collecting the produced polypeptide from the culture. Manufacturing method for the included polypeptide.  10. An antibody against the polypeptide according to any one of claims 1 to 4 and 8.  11. The antibody according to claim 10, wherein the antibody is a polyclonal antibody.  12. The antibody according to claim 10, wherein the antibody is a monoclonal antibody.  13. A peptide fragment consisting of 10 or more consecutive amino acid residues in the amino acid sequence represented by any one of SEQ ID NOs: 4 to 10 in the sequence listing.  14. A method for enhancing gelatinase expression in mammalian skin cells, comprising using the polypeptide according to any one of claims 1 to 4 and 8.  15. A method for treating a wound, comprising using the polypeptide according to any one of claims 1 to 4 and 8. 16. A method for promoting the growth of mammalian hematopoietic cells, comprising using the polypeptide according to any one of claims 1 to 4 and 8.  17. A method for growing hematopoietic cells for bone marrow transplantation in vitro, comprising using the polypeptide described in any one of claims 1 to 4 and 8. 18. The anti-reflection device according to any one of claims 1 to 12 A method for suppressing overexpression of gelatinase in mammalian skin cells, comprising using a body.  19. A method for diagnosing or treating a disease associated with overexpression of gelatinase, comprising using the antibody according to any one of claims 10 to 12.  20. A method for suppressing hyperproliferation of hematopoietic cells, which comprises using the antibody according to any one of claims 10 to 12. 21. A method for diagnosing or treating a disease associated with hyperproliferation of hematopoietic cells, which comprises using the antibody according to any one of claims 10 to 12.  22. A composition comprising the polypeptide according to any one of claims 1 to 4 and 8 as an active ingredient. 23. A composition comprising the antibody according to any one of claims 10 to 12 as an active ingredient. 24. The composition according to claim 22 or 23 in the form of a cosmetic, pharmaceutical or reagent.  25. The composition according to claim 22 as a gelatinase expression enhancer.  26. The composition according to claim 22 as a wound healing promoter.  27. The composition according to claim 22 as a hematopoietic cell proliferation promoter.  28. A susceptible disease agent comprising the antibody according to any one of claims 10 to 12 as an active ingredient. 29. The susceptible disease agent according to claim 28, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody. 30. The susceptible disease agent according to claim 28 or 29, which is an angiogenesis inhibitor.  31. The agent for susceptibility disease according to claim 28 or 29 as a sebum production inhibitor.