JP2006212033A - Differentiation-suppressive polypeptide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel factor which is important for regeneration therapy and has a differentiation-suppressive activity to undifferentiated cells, and an effective medicine useful for tissue regeneration and proliferation maintenance of undifferentiated cells using the same. <P>SOLUTION: Human Delta-1 and Serrate-1 peptides which are ligands of a Notch protein controlling the proliferation of cerebral nerve, muscle and blood cell systems to human homologue and have a specific amino acid sequence are produced by genetic recombination of animal cells, and differentiation-suppressive agents for undifferentiated cells and media for cell culture are obtained thereby. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel physiologically active substance for suppressing differentiation of undifferentiated cells.

  There are many types of cells in human blood and lymph, and each plays an important role. For example, red blood cells protect the body from oxygen transport, platelets protect against hemostasis, and white blood cells and lymphocytes protect against infection. These diverse cells are derived from hematopoietic stem cells in the bone marrow. It has recently been revealed that hematopoietic stem cells are stimulated by various cytokines and environmental factors in the body and differentiate into various blood cells, osteoclasts, mast cells and the like. As this cytokine, erythropoietin (EPO) is used for differentiation into red blood cells, granulocyte colony-stimulating factor (G-CSF) is used for differentiation into white blood cells, and platelet growth factor (G-CSF) is used for differentiation into megakaryocytes, which are platelet-producing cells. mpl ligand) was discovered, and the former two are already in clinical application.

  Blood undifferentiated cells are conceptually classified into blood progenitor cells destined to differentiate into a specific blood lineage and hematopoietic stem cells having the ability to differentiate into all lineages and self-replicating ability. Although it is possible to identify blood progenitor cells by colony assay, a method for identifying hematopoietic stem cells has not been established. Regarding these cells, stem cell factor (SCF), interleukin 3 (IL-3), granulocyte monocyte colony stimulating factor (GM-CSF), interleukin 6 (IL-6), interleukin 1 (IL-1) It has been reported that granulocyte colony-stimulating factor (G-CSF), oncostatin M and the like promote cell differentiation and proliferation. In order to apply hematopoietic stem cell transplantation therapy and gene therapy as an alternative to bone marrow transplantation therapy, it has been studied to amplify hematopoietic stem cells in vitro. However, when these cells are grown and cultured in vitro using cytokines as described above, the pluripotency and self-replicating ability inherent in hematopoietic stem cells are gradually lost, and after 5 weeks of culture, the cells become a specific lineage. It has been reported that pluripotency, which is one of the characteristics of hematopoietic stem cells, is lost (Rice et al., Blood 86, 512-523, 1995).

  It has been clarified that a single cytokine alone is not sufficient for the proliferation of blood progenitor cells, and the joint action (synergy) of multiple cytokines is important. Therefore, in order to proliferate while maintaining the characteristics of hematopoietic stem cells, it is considered that a cytokine that suppresses differentiation is required together with a cytokine that proliferates and differentiates blood undifferentiated cells. However, in general, many cytokines that promote cell proliferation and differentiation are found, whereas only a few cytokines suppress cell differentiation. For example, leukemia cell inhibitory factor (LIF) has been reported to proliferate mouse embryonic stem cells without differentiation, but has no such effect on hematopoietic stem cells and blood progenitor cells. Moreover, although tumor cell growth factor (TGF-β) acts to suppress the growth of various cells, a certain opinion has not been obtained on the effects on hematopoietic stem cells and blood progenitor cells.

  It is considered that not only blood cells but also undifferentiated cells, especially stem cells, are strongly involved in tissue regeneration. Regenerating these tissues and amplifying the undifferentiated cells of each tissue can be known for a wide range of uses by referring to Seisho (Katsuri Yoshizato Regenerative Structure, 1996, Yodosha).

  Notch is a receptor-type membrane protein involved in neuronal differentiation control discovered in Drosophila. Notch homologs are nematodes (Lin-12), Xenopus (Xotch), mice (Motch), humans (Motch) It has been found from a wide range of animal species beyond the classification of invertebrates and vertebrates such as TAN-1). On the other hand, two Drosophila Notch ligands, Drosophila Delta (Delta) and Drosophila Serrate, have been found, and notch ligand homologs have been found from a wide range of animal species, similar to the receptor Notch (Artavanis-Tsakonas etal). Science, 268, 225-232, 1995).

  Especially for humans, the human Notch homolog TAN-1 is widely expressed in tissues throughout the body (Ellisen et al., Cell 66, 649-661, 1991), and there are two notch analogs besides TAN-1. The presence of molecules has been reported (Artavanis-Tsakonas et al., Science 268, 225-232, 1995). In blood cells, expression of TAN-1 was observed in CD34 positive cells by PCR (Polymerase Chain Reaction) method (Milner et al., Blood 83, 2057-2062, 1994). However, regarding humans, there has been no report on cloning of human delta and human selenate genes that are considered ligands for Notch.

The Drosophila Notch has been examined in detail for its binding to the ligand. Among the Epidermal Growth Factor (EGF) -like repetitive amino acid sequences of 36 in the extracellular part of the Notch, the 11th and 12th repetitive amino acid sequences are used as the binding regions, and the ligand is used. And Ca ⁺⁺ (Fehon et al., Cell 61, 523-534, 1990 and Rebay et al., Cell 67, 687-699, 1991 and JP 7). -503123). The EGF repeat sequences are also conserved in other types of Notch homologues, and a similar mechanism is inferred for binding to the ligand. Also in the ligand, an amino acid sequence called DSL (Delta-Serrate-Lag-2) and an EGF-like repetitive sequence are conserved in the vicinity of the amino acid terminal (Artavanis-Tsakonas et al., Science 268, 225-232). , 1995).

  No such sequence has been found in the DSL domain other than the Notch ligand molecule, and the DSL domain has a structure unique to the Notch ligand molecule. The consensus sequence of this DSL domain is represented by SEQ ID NO: 1 in the sequence listing as a general formula, and FIG. 1 shows a comparison between human delta-1, human serato 1 and other known Notch ligand molecules of the present invention.

  On the other hand, EGF-like sequences include thrombomodulin (Jackman et al., Proc. Natl. Acad. Sci. USA 83, 8834-8838, 1986) and low density lipoprotein (LDL) receptor (Russell et al., Cell 37, 577- 585, 1984) and blood coagulation factors (Furie et al., Cell 53, 505-518, 1989), and are thought to play an important role in extracellular aggregation and adhesion.

  In recent years, cloned Drosophila delta vertebrate homologues have been found in chickens (C-delta-1) and Xenopus (X-delta-1), and X-delta-1 adds Xotch to the development of primitive neurons. (Henrique et al., Nature 375,787-790, 1995 and Chitnis et al., Nature 375, 761-766, 1995). On the other hand, as a vertebrate homologue of Drosophila seleate, rat jagged is found (Claire et al., Cell 80, 909-917, 1995). According to this report, rat jagged mRNA is detected in the spinal cord of fetal rats. In addition, co-culture of a myoblast cell line that forcibly overexpressed rat notch and a rat jagd-expressing cell line has been found to suppress the differentiation of this myoblast cell line. It has been found that rat jagged does not act on myoblast cell lines.

  From these reports, Notch and its ligands are thought to be involved in the regulation of neuronal differentiation. However, some cells, except for myoblasts, may be used as primary cells. The effect was completely unknown.

  In addition, the Notch ligand molecule is characterized by having a DSL domain structure that has not been found at least other than the Notch ligand molecule from studies of these Drosophila and nematodes. Therefore, having this DSL domain may be considered equivalent to being a ligand molecule for the Notch receptor.

Rice et al. , Blood 86, 512-523, 1995 Yoshisato Victory Regeneration-How to Talk, 1996, Yodosha Artavanis-Tsakonas et al. , Science 268, 225-232, 1995 Ellisen et al. Cell 66, 649-661, 1991. Artavanis-Tsakonas et al. , Science 268, 225-232, 1995 Milner et al. , Blood 83, 2057-2062, 1994 Fehon et al. , Cell 61, 523-534, 1990 Rebay et al. Cell 67, 687-699, 1991. Jackman et al. , Proc. Natl. Acad. Sci. USA 83, 8834-8838, 1986 Russell et al. , Cell 37, 577-585, 1984 Furie et al. , Cell 53, 505-518, 1989. Henrique et al. , Nature 375, 787-790, 1995. Chitnis et al. , Nature 375, 761-766, 1995 Claire et al. , Cell 80, 909-917, 1995 Special table flat 7-503123

  As described above, a method for growing undifferentiated cells while maintaining their characteristics has not been completed. The main cause of this is that sufficient factors that suppress the differentiation of undifferentiated cells have not been found. An object of the present invention is to provide a compound derived from a novel factor that suppresses differentiation of undifferentiated cells.

  The present inventors hypothesized that Notch and its ligand not only control differentiation of neuroblasts and myoblasts but also control differentiation of widely undifferentiated cells, particularly blood undifferentiated cells. However, when clinically applied to humans, known Notch ligands of different species such as chicken and Xenopus have species specificity and antigenicity problems. For this reason, it is indispensable to obtain a human-type Notch ligand that has not yet been reported. Therefore, the present inventors are molecules having a DSL domain and an EGF-like domain common to Notch ligand molecules, and human delta homolog (hereinafter referred to as human delta) and human serate homolog (hereinafter referred to as human delta) ligands of human type Notch (TAN-1 etc.). The human serites were considered to exist, and these findings were considered to be candidates for effective drugs for controlling the differentiation of undifferentiated cells.

  The present inventors analyzed the conserved amino acid sequence of these homologs found in non-human animals for the search for human Notch ligands, and found this gene by PCR with mixed primers of the corresponding DNA sequence. Proceeded as much as possible. As a result of intensive studies, the inventors succeeded in isolating cDNAs encoding the amino acid sequences of two novel molecules having a DSL domain common to Notch ligand molecules, novel human delta-1 and novel human seratol. Using this, protein expression systems having various forms were prepared. In addition, a purification method for these proteins was established, purified and isolated.

  The amino acid sequence of the novel human delta-1 is shown in SEQ ID NO: 2 to 4 in the sequence listing, and the DNA sequence encoding them is shown in SEQ ID NO: 8 in the sequence listing. The amino acid sequence of novel human serate-1 is shown in SEQ ID NO: 5 to 7 in the sequence listing, and the DNA sequence encoding them is shown in SEQ ID NO: 9 in the sequence listing.

  Physiological action of the protein produced in this way can be performed using a large number of cells such as neuronal undifferentiated cells, preadipocytes, hepatocytes, myoblasts, skin undifferentiated cells, blood undifferentiated cells and immune undifferentiated cells. Explored. As a result, it was found that the novel human delta-1 and the novel human selete-1 have a differentiation control action on primary blood undifferentiated cells and have a physiological action to maintain the undifferentiated state.

  Such an action on blood undifferentiated cells has not been reported in the past, and is a completely new finding. Furthermore, in the toxicity test for mice, no obvious toxicity was observed, and the effect of becoming an effective pharmaceutical product was shown, and the present invention was completed. Therefore, the drug containing the molecule of the present invention, the medium containing the molecule of the present invention, and the device on which the molecule of the present invention is immobilized are completely new drugs and medical products that can keep blood undifferentiated cells in an undifferentiated state. Further, antibodies against each of the human delta-1 and the human serate-1 were used as immunogens, purification methods were established, and the present invention was completed.

  That is, the present invention relates to a polypeptide containing the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing encoded by a human-derived gene, a polypeptide containing at least the amino acid sequence set forth in SEQ ID NO: 2 or 5 in the sequence listing, A polypeptide containing the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing, a polypeptide containing the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing, a polypeptide containing the amino acid sequence shown in SEQ ID NO: 6 in the sequence listing, A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 7 in the sequence listing, a polypeptide having an action of inhibiting differentiation of undifferentiated cells, a polypeptide in which the undifferentiated cells are undifferentiated cells other than cranial nerves, muscle undifferentiated cells, The present invention relates to a polypeptide in which the undifferentiated cell is a blood undifferentiated cell, and the present invention relates to a pharmaceutical composition containing the above polypeptide, and its use The present invention relates to a cell culture medium containing the above polypeptide, a cell culture medium in which the cells are blood undifferentiated cells, and the present invention further relates to at least SEQ ID NO: 2 in the sequence listing. Alternatively, a DNA encoding a polypeptide containing the amino acid sequence described in 5, a DNA sequence from No. 242 to 841 of a DNA sequence shown in SEQ ID NO: 8 of the sequence listing or a DNA sequence shown in SEQ ID NO: 9 of the sequence listing DNA containing the DNA sequence of Nos. 502 to 1095, DNA encoding the polypeptide containing the amino acid sequence of SEQ ID No. 3 of the sequence listing, and DNA sequences No. 242 to 1801 of the DNA sequence of SEQ ID No. 8 of the sequence listing A DNA encoding the polypeptide containing the amino acid sequence of SEQ ID NO: 4 in the sequence listing DNA containing the DNA sequence of Nos. 242 to 2347 of the DNA sequence shown in SEQ ID NO: 8 of the Sequence Listing, DNA encoding the polypeptide containing the amino acid sequence of SEQ ID NO: 6 of Sequence Listing, DNA containing the DNA sequence of Nos. 502 to 3609 of the DNA sequence of SEQ ID NO: 9, DNA encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 7 of the sequence listing, and SEQ ID NO: 9 of the sequence listing DNA comprising the DNA sequence of Nos. 502 to 4062 of the described DNA sequence, and the present invention is a recombinant DNA obtained by ligating a DNA selected from the above DNA group and a vector DNA that can be expressed in a host cell Body, cells transformed with recombinant DNA body, and method for producing polypeptide by culturing cells and collecting compound produced from culture The present invention relates to an antibody that specifically recognizes a polypeptide having the amino acid sequence of SEQ ID NO: 4 in the sequence listing, and an antibody that specifically recognizes a polypeptide having the amino acid sequence of SEQ ID NO: 7 in the sequence listing.

  The Notch ligand molecule of the present invention is an effective chemical product for maintaining undifferentiated cells and inhibiting differentiation, and can be used as a pharmaceutical product or a medical product.

Hereinafter, the present invention will be described in detail.
A series of molecular biological experiments such as preparation of cDNA necessary for gene manipulation, examination of expression by Northern blot, screening by hybridization, preparation of recombinant DNA, determination of DNA base sequence, preparation of cDNA library, etc. It can be performed by the method described in a normal experiment document. Examples of the above-mentioned normal experimental documents include, for example, Molecular Cloning edited by Maniatis et al., A laboratory manual, 1989, Eds. Sambrook, J .; , Fritsch, E .; F. , And Maniatis, T .; , Cold Spring Harbor Laboratory Press.

  The polypeptide of the present invention has a polypeptide consisting of at least the amino acid sequences of SEQ ID NOs: 1 to 7 in the sequence listing, but is known to occur in nature, such as intraspecies variation, allelic variation, and the like, and artificially Variants produced by mutations that can be made are included in the novel polypeptides of the present invention as long as the polypeptides of SEQ ID NOs: 1 to 7 in the Sequence Listing do not lose their properties. The amino acid modification and substitution are described in detail, for example, in an application by Benntt et al. (International Publication No. WO96 / 2645) and can be prepared with reference to these.

  In addition, a DNA sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 to 4 in the sequence listing encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 5 to 7 in the sequence listing for SEQ ID NO: 8 in the sequence listing. The DNA sequence is shown together with the amino acid sequence in SEQ ID NO: 9 in the sequence listing. With respect to these gene sequences, even if there is no mutation at the amino acid level, in the chromosomal DNA or cDNA isolated from nature, the base sequence of the DNA can be changed without changing the amino acid sequence encoded by the DNA due to the degeneracy of the genetic code. Mutated cases are often seen. Further, since the 5 ′ untranslated region and the 3 ′ untranslated region are not involved in the definition of the amino acid sequence of the polypeptide, the DNA sequence in these regions is easily mutated. The base sequence obtained by such degeneracy of the genetic code is also included in the DNA of the present invention.

  In the present invention, an undifferentiated cell is defined as a cell that can be proliferated by a specific stimulus and that can differentiate into a cell having a specific function by a specific stimulus. Differentiated cells, undifferentiated cells of the cranial nervous system, undifferentiated cells of the muscular system, undifferentiated cells of the blood system, etc., each having a self-replicating ability called a stem cell and capable of generating cells of that lineage Including. Further, the differentiation inhibitory action is an action that suppresses the phenomenon that these undifferentiated cells differentiate autonomously or irregularly, and specifically, an action that maintains an undifferentiated state. In addition, the cranial nervous system undifferentiated cells can be defined as cells having the ability to differentiate only into brain and nerve cells having a specific function in accordance with a specific stimulus. In addition, muscular undifferentiated cells are defined as cells having the ability to differentiate only into muscle cells having a specific function with a specific stimulus. The blood undifferentiated cells described in the present invention are blood progenitor cells destined to differentiate into a specific blood line that can be identified by a blood colony assay, and the ability to differentiate into all lineages and self-replicating ability. Is defined as a cell group containing hematopoietic stem cells having

  In the sequence listing, the amino acid sequence of SEQ ID NO: 1 shows a common amino acid sequence of a DSL domain, which is a domain structure common to Notch ligand molecules, as a general formula. At least the structure of this domain is related to human delta-1. It corresponds to 158 to 200 of SEQ ID NO: 2 in the sequence table, and corresponds to 156 to 198 of SEQ ID NO: 5 in the sequence listing for human serate-1.

  The amino acid sequence of SEQ ID NO: 2 in the sequence listing is the active center sequence excluding the human delta-1 signal peptide of the present invention, that is, the amino acid sequence from the amino terminus to the DSL domain, and the human delta of the present invention shown in SEQ ID NO: 4 Corresponds to amino acid numbers 1 to 200 of the mature full-length amino acid sequence of -1. The amino acid sequence of SEQ ID NO: 3 is the sequence of the extracellular domain excluding the human delta-1 signal peptide of the present invention, and amino acid number 1 of the human delta-1 mature full-length amino acid sequence of the present invention shown in SEQ ID NO: 4 To 520. The amino acid sequence of SEQ ID NO: 4 is the mature full-length amino acid sequence of human delta-1 of the present invention.

  The amino acid sequence of SEQ ID NO: 5 is the sequence of the active center excluding the signal peptide of human serate-1 of the present invention, that is, the amino acid sequence from the amino terminus to the DSL domain, and the human delta-1 of the present invention shown in SEQ ID NO: 7 Corresponds to amino acid numbers 1 to 198 of the mature full-length amino acid sequence. The amino acid sequence of SEQ ID NO: 6 is the sequence of the extracellular domain excluding the signal peptide of human serate-1 of the present invention, and amino acid number 1 of the mature full-length amino acid sequence of human serate-1 of the present invention shown in SEQ ID NO: 7 No. 1036. The amino acid sequence of SEQ ID NO: 7 is the mature full-length amino acid sequence of human serate-1 of the present invention.

  The sequence of SEQ ID NO: 8 is the entire amino acid sequence of human delta-1 of the present invention and the cDNA sequence encoding it. The sequence of SEQ ID NO: 9 of the sequence listing is the entire amino acid sequence of human serate-1 of the present invention and It is the cDNA sequence encoding it.

  The left end and the right end of the amino acid sequences described in the sequence listing are the amino group end (hereinafter referred to as N-terminal) and the carboxyl group end (hereinafter referred to as C-terminal), respectively, and the left end and right end of the base sequence are the 5 ′ end and 3 respectively. 'The end.

  The following method can be considered for cloning the gene of the human Notch ligand. Since a part of the amino acid sequence of human Notch ligand is conserved during the evolution of organisms, a DNA sequence corresponding to the conserved amino acid sequence is designed and used as a primer for RT-PCR (Reverse Transcription Polymerase Chain Reaction). There is a possibility that a human Notch ligand gene fragment can be obtained by amplifying a human-derived PCR template by PCR reaction. In principle, it is possible to prepare RT-PCR primers from the known DNA sequence information of Notch ligand homologues of organisms other than humans, and to obtain known gene fragments from the PCR templates of the organisms.

  To perform PCR for the purpose of obtaining human Notch ligand fragments, PCR for DSL sequences was first considered. However, the number of combinations of DNA sequences corresponding to amino acid sequences stored in this region is enormous, and PCR primer design is impossible. Therefore, EGF-like sequences had to be subjected to PCR. As described above, since EGF-like sequences are conserved in many molecules, fragment acquisition and identification are extremely difficult.

The present inventors designed and prepared about 50 sets of PCR primers using the sequence shown in Example 1 as an example, and performed PCR using cDNAs prepared from polyA ⁺ RNA of various human-derived tissues as PCR templates. This was carried out, and 10 or more kinds of PCR products were subcloned, and a total of 500 or more kinds of sequences were performed. As a result of diligent efforts, one kind of clone having the target sequence could be identified. That is, the obtained PCR product is cloned into a cloning vector, a host cell is transformed with a recombinant plasmid containing this PCR product, the host cell containing the recombinant plasmid is cultured in large quantities, and the recombinant plasmid is purified and purified. The DNA sequence of the PCR product inserted into the cloning vector was examined and compared with the amino acid sequence of another known delta for each, and efforts were made to obtain a gene fragment that appears to have the sequence of human delta-1. As a result, the inventors succeeded in finding a gene fragment containing the same sequence as DNA sequences Nos. 1012 to 1375 in the sequence listing, ie, a part of human delta-1 cDNA.

Similarly, the present inventors designed and prepared 50 sets of PCR primers using the sequence shown in Example 3 as an example, and cDNAs prepared from polyA ⁺ RNA of various human-derived tissues were used as PCR templates, respectively. PCR was carried out, 10 or more types of PCR products were subcloned, and a total of 500 or more sequences were performed. As a result of diligent efforts, one type of clone having the target sequence could be identified. That is, the obtained PCR product is cloned into a cloning vector, a host cell is transformed with a recombinant plasmid containing this PCR product, the host cell containing the recombinant plasmid is cultured in large quantities, and the recombinant plasmid is purified and purified. The DNA sequence of the PCR product inserted into the cloning vector was examined, and each was compared with the amino acid sequence of another known selete for each, and an effort was made to obtain a gene fragment that appears to have the human seleito 1 sequence. As a result, the inventors succeeded in finding a gene fragment containing a part of the DNA sequence shown in SEQ ID NO: 9 in the Sequence Listing, which is identical to the 1272th to 1737th DNA sequences, that is, a portion of the human Serato 1 cDNA.

Next, using the human delta-1 and human selete-1 gene fragments thus obtained, the full length of the target gene can be obtained from a human genomic gene library or cDNA library. Full-length cloning can be obtained by isotopic labeling the gene partially cloned by the above method and various non-isotopic labels, and screening the library by a method such as hybridization. As an isotope labeling method, for example, a method of labeling ends using [ ³² P] γ-ATP and T4 polynucleotide kinase, a labeling method by other nick translation methods or primer extension methods can be used. As another method, a cDNA library derived from a human can be incorporated into an expression vector, expressed in COS-7 cells, etc., and the cDNA of the ligand can be isolated by a technique such as expression cloning for screening the target gene. For expression cloning, a fractionation method using a cell sorter utilizing the binding of polypeptides containing amino acid sequences of four notches known to date such as TAN-1, a detection method using a film emulsion using a radioisotope, etc. A method is mentioned. Here, the method for obtaining the genes for human delta-1 and sateselate-1 has been described. However, it is useful to obtain the Notch ligand homologue genes of other organisms for the analysis of ligand action, and this can also be obtained by the same means. The obtained target gene can determine the DNA sequence and estimate the amino acid sequence.

  As shown in Example 2, the present inventors labeled a gene fragment containing a human delta-1 PCR product with a radioisotope, used as a hybridization probe, and performed screening using human placenta-derived cDNA as a screening library. When the DNA sequences of a plurality of clones were determined, it was revealed that the clone contained the DNA base sequence shown in SEQ ID NO: 8 in the sequence listing and encoded from the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing deduced therefrom. Thus, the cDNA encoding the full-length amino acid of human delta-1 was successfully cloned.

  When these sequences were compared in a database (Genbank release 89, June, 1995), they were novel sequences. According to the method of Kyte-Doolittle (J. Mol. Biol. 157: 105, 1982), the hydrophobic portion and the hydrophilic portion were analyzed from the amino acid sequence. As a result, it was revealed that human delta-1 of the present invention is expressed on cells as a cell membrane protein having one cell membrane passage portion.

  Similarly, as shown in Example 4, the present inventors labeled a gene fragment containing a human serate-1 PCR product with a radioisotope, used as a hybridization probe, and screened using human placenta-derived cDNA as a screening library, When the DNA sequences of the obtained plurality of clones were determined, it was a clone containing the DNA base sequence shown in SEQ ID NO: 9 in the sequence listing, and a part of the amino acid sequence shown in SEQ ID NO: 7 in the sequence listing deduced therefrom was obtained. It became clear to code. Since part of the intracellular portion of the gene sequence encoding the full-length amino acid sequence, that is, the portion corresponding to the vicinity of the stop codon could not be cloned in this screening, as shown in Example 4, the RACE method (rapid amplification of cDNA ends method, Frohman et al., Proc. Natl. Acad. Sci. USA 85, 8998-9002, 1988) and finally cloning cDNA encoding the full-length amino acid of human serate-1. Successful.

  When this sequence was compared in a database (Genbank release 89, June, 1995), they were novel sequences. According to the method of Kyte-Doolittle (J. Mol. Biol. 157: 105, 1982), the hydrophobic portion and the hydrophilic portion were analyzed from the amino acid sequence. As a result, it was revealed that human serate-1 of the present invention is expressed on cells as a cell membrane protein having one cell membrane passage portion.

  Examples of the plasmid into which cDNA is incorporated include pBR322, pUC18, pUC19, pUC118, and pUC119 (all manufactured by Takara Shuzo Co., Ltd., Japan) derived from Escherichia coli. Any of them can be used. Examples of phage vectors into which cDNA is incorporated include λgt10, λgt11, and the like, but other types can be used as long as they can be propagated in the host. Thus, the obtained plasmid is introduce | transduced into a suitable host, for example, Escherichia genus bacteria, Bacillus genus bacteria, etc. using the calcium chloride method etc. Examples of the genus Escherichia include Escherichia coli K12HB101, MC1061, LE392, JM109, and the like. Examples of the Bacillus genus include Bacillus and Sachiris MI114. In addition, the phage vector can be introduced into, for example, grown Escherichia coli using an in vitro packaging method (Proc. Natl. Acad. Sci., 71: 2442-, 1978).

  According to the analysis of the amino acid sequence of human delta-1, the amino acid sequence of the precursor of human delta-1 is composed of 723 amino acid residues shown in the amino acid sequence of SEQ ID NO: 8 in the sequence listing, and the signal peptide region is the amino acid sequence of the sequence listing. 21 amino acid residues corresponding to serine from -21 of -21 (SEQ ID NO: 8) to -1 (21 of SEQ ID NO: 8) serine, the extracellular region is 1 of the sequence listing (22 of SEQ ID NO: 8) No.) Serine to No. 520 (No. 541 of SEQ ID No. 8) Glycine, 520 amino acid residues, and the cell membrane passage region is the amino acid sequence of No. 521 (No. 542 of SEQ ID No. 8) of the same sequence listing from Proline No. 552 (sequence) 32 amino acid residues corresponding to leucine of No. 8 (573), and the intracellular region is from the glutamine of No. 553 (No. 574 of SEQ ID No. 8) to 702 (sequence) of the same sequence listing It has been estimated that 150 amino acid residues corresponding to valine 723 number) of issue 8 corresponds. However, each of these parts is a domain structure predicted from the amino acid sequence to the last, and it is considered that the existence form in cells and in solution is slightly different from the above structure. It is also conceivable that the constituent amino acids of each domain are around 5 to 10 amino acid sequences.

  Comparison of amino acid sequences with other delta homologues of human delta-1 revealed that the homologies with Drosophila delta, chicken delta and Xenopus delta were 47.6%, 83.3% and 76.2%, respectively. This is a novel substance having a novel amino acid sequence different from that of the above substance, and is a substance that has been revealed for the first time by the present inventors. In addition, no polypeptide having the same sequence as human delta-1 was found in the search for all species on the database.

  Notch's ligand homologues have an evolutionarily conserved common sequence. That is, it is an EGF-like sequence that repeats with the DSL sequence. These conserved sequences were deduced from the amino acid sequence of human delta-1 by comparison of human delta-1 with delta homologs of non-human species. That is, the DSL sequence corresponded to 43 amino acid residues corresponding to cysteine from 158 to 200 in the amino acid sequence of SEQ ID NO: 4 in the Sequence Listing. The EGF-like sequence is repeated 8 times. Among the amino acid sequences of SEQ ID NO: 4 in the sequence listing, the first EGF-like sequence is from 205 to 233 cysteine, and the second EGF-like sequence is from 236 to 264 cysteine. The 3rd EGF-like sequence is from 271 to 304, the 4th EGF-like sequence is from 311 to 342, the 5th EGF-like sequence is from 349 to 381, and the 6th EGF-like sequence is 388. From cysteine to 419, the 7th EGF-like sequence corresponds to cysteine from 426 to 457, and the 8th EGF-like sequence corresponds to 464 to 495.

  As expected from the amino acid sequence of human delta-1, the portion to which a sugar chain is added is the portion where N-acetyl-D-glucosamine can bind to N-glucoside, and the number 456 of the amino acid sequence of SEQ ID NO: 4 in the sequence listing. Asparagine residues. Moreover, as a part which estimates the O-glycoside bond of N-acetyl-D-galactosamine, the part where a serine or threonine residue appears frequently is considered. Proteins to which these sugar chains have been added are generally more stable against degradation in vivo than polypeptides themselves, and are considered to have strong physiological activity. Accordingly, N-acetyl-D-glucosamine is an N-glucoside or N-acetyl-D-galactosamine sugar chain such that N-acetyl-D-glucosamine is included in the amino acid sequence of a polypeptide containing the sequence of SEQ ID NO: 2, 3, or 4 in the sequence listing. Polypeptides formed by -glucoside or O-glucoside linkage are also included in the present invention.

  Further, according to the analysis of the amino acid sequence of human serate-1, the amino acid sequence of the precursor of human serate-1 is composed of 1218 amino acid residues shown in the amino acid sequence of SEQ ID NO: 9 in the sequence listing, and the signal peptide region is shown in the sequence listing. 31 amino acid residues from the methionine of amino acid sequence -31 (No. 1 of SEQ ID NO: 9) to alanine of -1 (No. 31 of SEQ ID NO: 9), the extracellular region is No. 1 (SEQ ID NO: 9) No. 32) serine to 1036 amino acid residues (1067 of SEQ ID NO: 9) aspartic acid, 1036 amino acid residues, and the cell membrane passage region is 1062 from phenylalanine of amino acid sequence 1037 (SEQ ID NO: 9 1068) of the same sequence listing. 26 amino acid residues corresponding to leucine of No. (SEQ ID NO: 91093), intracellular region is 1063 (SEQ ID NO: 9 of the same sequence listing) 106 the amino acid residue corresponding to valine 094 No.) No. 1187 from arginine (1218 of SEQ ID NO: 9) was estimated to be appropriate. However, each of these parts is a domain structure predicted from the amino acid sequence to the last, and it is considered that the existence form in cells and in solution is slightly different from the above structure. It is also conceivable that the constituent amino acids of each domain are around 5 to 10 amino acid sequences.

  Comparing the amino acid sequence of human serate-1 with serate homologues of other organisms, the homology with Drosophila selete and rat jagd was 32.1% and 95.3%, respectively. It is a novel substance having a sequence, and is a substance that has been revealed for the first time by the present inventors. In addition, no polypeptide having the same sequence as human selete-1 was found in the search for all species on the database.

  Notch's ligand homologues have an evolutionarily conserved common sequence. That is, it is an EGF-like sequence that repeats with the DSL sequence. These conserved sequences were deduced from the amino acid sequence of human serate-1 by comparing human serate-1 with other Notch ligand homologs. That is, the DSL sequence corresponded to 43 amino acid residues corresponding to cysteine from 156 to 198 in the amino acid sequence of SEQ ID NO: 7 in the Sequence Listing. The EGF-like sequence is repeated 16 times. Among the amino acid sequences of SEQ ID NO: 7 in the sequence listing, the first EGF-like sequence is from cysteine 204 to 231 cysteine, and the second EGF-like sequence is from 234 to 262 cysteine. The 3rd EGF-like sequence is from 269 to cysteine, the 4th EGF-like sequence is from 309 to 340 cysteine, the 5th EGF-like sequence is from 346 to 378 cysteine, and the 6th EGF-like sequence is 385. From cysteine to 416 cysteine, 7th EGF-like sequence from 423 cysteine to 453 cysteine, 8th EGF-like sequence from 460 cysteine to 491 cysteine, 9th EGF-like sequence from 498 cysteine to 529 cysteine, 10EGF Sequences from 536 cysteine to 595 cysteine, 11th EGF-like sequence from 602 to 633 cysteine, 12th EGF-like sequence from 640 to 671 cysteine, 13th EGF-like sequence from 678 to 709 Up to cysteine, the 14th EGF-like sequence corresponds to cysteine from 717 to 748, the 15th EGF-like sequence from 755 to 786, and the 16th EGF-like sequence from 793 to 824. However, the 10th EGF-like sequence had an irregular sequence containing 10 residues of cysteine.

  As expected from the amino acid sequence of human serato 1, the portion to which a sugar chain is added is the portion where N-acetyl-D-glucosamine can bind to N-glucoside, the 112th amino acid sequence of SEQ ID NO: 7 in the sequence listing, 131, 186, 351, 528, 554, 714, 1014, 1033 asparagine residues. Moreover, as a part which estimates the O-glycoside bond of N-acetyl-D-galactosamine, the part where a serine or threonine residue appears frequently is considered. Proteins to which these sugar chains have been added are generally more stable against degradation in vivo than polypeptides themselves, and are considered to have strong physiological activity. Therefore, N-acetyl-D-glucosamine is an N-glucoside or N-acetyl-D-galactosamine sugar chain such as N in the amino acid sequence of the polypeptide containing the sequence number 5, 6 or 7 in the sequence listing. Polypeptides formed by -glucoside or O-glucoside linkage are also included in the present invention.

  Studies on the binding of Drosophila Notch and its ligand reveal that the amino acid region required for the Drosophila Notch ligand to bind to Notch is from the N-terminal to the DSL sequence of the mature protein from which the signal peptide was cleaved. (Special Table No. 7-503121). From this, the region necessary for the expression of the ligand action of the human Notch ligand molecule is at least the DSL domain, that is, the region containing the amino acid sequence of SEQ ID NO: 1 in the sequence listing, and at least necessary for the expression of the ligand action of human delta-1. It can be seen that the region is the novel amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and that at least the region necessary for the expression of the ligand action of human selete-1 is the novel amino acid sequence shown in SEQ ID NO: 5 in the sequence listing.

  Further, if DNA encoding a part or all of the gene sequence of SEQ ID NO: 8 in the sequence listing is used, it is possible to detect human delta-1 mRNA, and a part of the gene sequence of SEQ ID NO: 9 of the sequence listing or If DNA encoding all of them is used, it is possible to detect human Serte-1 mRNA. For example, as a method for examining the expression of these genes, nucleic acids capable of complementing 12 mer to 16 mer or more, more preferably 18 mer or more having a partial gene sequence of SEQ ID NO: 8 or 9 in the sequence listing, that is, antisense DNA, RNA , And derivatives in which they are methylated, methylphosphated, deaminated, or thiophosphated, and can be performed by techniques such as hybridization and PCR. The homologue of this gene from other organisms such as mice and gene cloning can be detected by the same method. Furthermore, cloning of genes on the genome including humans is possible as well. Therefore, by using these genes cloned in such a manner, more detailed functions of the present human delta-1 and human serate-1 can be clarified. For example, if a recent genetic manipulation technique is used, any method such as a transgenic mouse, a gene targeting mouse, or a double knockout in which both genes related to the present gene are inactivated can be used. Moreover, if there is an abnormality in the genome of this gene, it can be applied to gene diagnosis and gene therapy.

  A transformed cell in which the vector pUCDL-1F containing cDNA encoding the entire amino acid sequence of human delta-1 of the present invention was introduced into E. coli JM109 was obtained by using E. coli. Eli: JM109-pUCDL-1F has been deposited with the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, located in 1-3 1-3 Higashi, Tsukuba, Ibaraki, Japan. The deposit date is October 28, 1996, and the deposit number is FERM BP-5728. In addition, a transformed cell obtained by introducing a vector pUCSR-1 containing cDNA encoding the entire amino acid sequence of human serate-1 of the present invention into E. coli JM109 is E. coli. Eli: JM109-pUCSR-1 has been deposited with the Institute of Biotechnology, Institute of Industrial Technology, Ministry of International Trade and Industry located in 1-3-3 Higashi, Tsukuba City, Ibaraki, Japan. The deposit date is October 28, 1996, and the deposit number is FERM BP-5726.

  Numerous methods are available for the expression and purification of human delta-1 and human serate-1 having various forms using cDNA encoding the amino acid sequences of human delta-1 and human serate-1 separated by the above method. (Kriegler, Gene Transfer and Expression-A Laboratory Manual Stockton Pres, 1990, and Yokota et al., BioManual Series 4, Gene Introduction and Expression / Analysis Methods, Yodosha, 1994). That is, the isolated cDNA encoding the human delta-1 and human serate-1 amino acid sequences is connected to an appropriate expression vector to produce eukaryotic cells such as animal cells and insect cells, and prokaryotic cells such as bacteria. Can do.

  When expressing human delta-1 and human serate-1 of the present invention, the DNA encoding the polypeptide of the present invention has a translation initiation codon at the 5 ′ end and a translation stop codon at the 3 ′ end. May be. These translation initiation codon and translation termination codon can also be added using an appropriate synthetic DNA adapter. Further, a promoter is connected upstream to express the DNA. Examples of the vector include the aforementioned E. coli-derived plasmid, Bacillus subtilis-derived plasmid, yeast-derived plasmid, bacteriophage such as λ phage, and animal viruses such as retrovirus and vaccinia virus.

  The promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression.

  When the host for transformation is Escherichia, tac promoter, trp promoter, lac promoter and the like are preferable. When the host is Bacillus, SPO1 promoter, SPO2 promoter and the like are preferable, and the host is yeast. In some cases, PGK promoter, GAP promoter, ADH promoter and the like are preferable.

  When the host is an animal cell, an SV40-derived promoter such as the SRα promoter described in the Examples, a retrovirus promoter, a metal thionein promoter, a heat shock promoter, or the like can be used.

  By using an expression vector having a promoter that can be used by any person skilled in the art as described above, the polypeptide of the present invention can be expressed.

  When expressing the polypeptide of the present invention, DNA encoding the amino acid sequence of SEQ ID NO: 2, 3, 4, 5, 6 or 7 in the sequence listing may be used alone, but it facilitates detection of the produced polypeptide. Therefore, a protein having a special function can be produced by adding a cDNA encoding a known antigen epitope for adding a cDNA encoding an immunoglobulin Fc to form a multimeric structure.

As for human delta-1, as shown in Example 5, the present inventors expressed as an expression vector expressing an extracellular protein,
1) DNA encoding amino acids 1 to 520 of the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing;
2) 8 amino acids at the C-terminal side of amino acids 1 to 520 of the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing, that is, Asp Tyr Lys Asp Asp Asp Asp Lys amino acid sequence (hereinafter referred to as FLAG sequence, sequence listing sequence) DNA encoding a chimeric protein to which a polypeptide having number 10) is added, and 3) the hinge part of human IgG1 below the C-terminal side of amino acids 1 to 520 of the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing Fc sequence (see International Publication No. WO94 / 02035) is added, and a DNA encoding a chimeric protein having a dimer structure is formed by disulfide bond at the hinge portion. Expression vector pMKITNeo (Maruyama et al. Proceedings, available from Maruyama, Tokyo Medical and Dental University, Japan Connecting each separately have a SR alpha), was produced extracellular portions expression vector of human Delta-1.

In addition, as an expression vector for expressing the full-length protein of human delta-1,
4) DNA encoding amino acids 1 to 702 of SEQ ID NO: 4 in the sequence listing, and 5) polypeptide having a FLAG sequence on the C-terminal side of amino acids 1 to 702 of SEQ ID NO: 4 in the sequence listing The DNA encoding the added chimeric protein was separately ligated to the expression vector pMKITNeo to prepare a full-length expression vector for human delta-1. A transformant is produced using the expression plasmid containing the DNA encoding the human delta-1 thus constructed.

Similarly, as shown in Example 6 for human selete-1, the present inventors expressed an expression vector for expressing an extracellular protein.
6) DNA encoding amino acids 1 to 1036 of the amino acid sequence set forth in SEQ ID NO: 6 in the sequence listing;
7) DNA encoding a chimeric protein obtained by adding a polypeptide having the FLAG sequence to the C-terminal side of amino acids 1 to 1036 of the amino acid sequence shown in SEQ ID NO: 6 in the sequence listing; and 8) Sequences of the sequence listing The Fc sequence is added to the C-terminal side of amino acids 1 to 1036 of the amino acid sequence shown in No. 6, and a DNA encoding a chimeric protein having a dimer structure is added to the expression vector pMKITNeo by a disulfide bond at the hinge portion. Each was ligated separately to produce an extracellular part expression vector for human seroto1.

In addition, as an expression vector for expressing the full-length protein of human selete-1,
9) DNA encoding amino acids 1 to 1187 of SEQ ID NO: 7 in the sequence listing, and 10) Polypeptide having a FLAG sequence on the C-terminal side of amino acids 1 to 1187 of SEQ ID NO: 7 in the sequence listing The DNA encoding the added chimeric protein was separately ligated to the expression vector pMKITNeo to prepare a full-length expression vector for human serate-1. A transformant is produced using the expression plasmid containing the DNA encoding human selate-1 constructed as described above.

  Examples of the host include Escherichia bacteria, Bacillus bacteria, yeast, animal cells, and the like. Examples of animal cells include monkey cells such as COS-7, Vero, Chinese hamster cell CHO, and silkworm cell SF9.

  As shown in Example 7, each of the above expression vectors 1) to 10) was separately transfected, and human delta-1 or human serate-1 was obtained from COS-7 cells (available from RIKEN, Cell Development Bank, RCB0539) and transformants transformed with these expression plasmids are obtained. Furthermore, various human delta-1 polypeptides and human selete-1 polypeptides can be produced by culturing each transformant by a known method in an appropriate medium under an appropriate culture condition.

  As shown in Example 8, human delta-1 polypeptide and human selete-1 polypeptide can be separated and purified from the culture as described above. Moreover, generally it can carry out by the following method.

  That is, when extracting from cultured microbial cells or cells, after culturing, the microbial cells or cells are collected by a known method, such as centrifugation, and suspended in an appropriate buffer, and subjected to ultrasound, lysozyme and / or A method of obtaining a crude extract by centrifugation or filtration after disrupting cells or cells by freeze-thawing or the like can be appropriately used. The buffer solution may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100. When secreted into the culture solution, the culture solution is separated from the cells or cells by a known method such as centrifugation, and the supernatant is collected.

  The human delta-1 and human selete-1 contained in the cell extract or cell supernatant thus obtained can be purified by using a known protein purification method. In order to confirm the presence of the protein during the purification process, in the case of the above-mentioned fusion proteins such as FLAG and human IgG Fc, it is possible to proceed with purification by detecting by immunoassay using antibodies against these known antigenic epitopes. it can. Moreover, when not expressing as such a fusion protein, it can detect using the antibody described in Example 9.

  Antibodies specifically recognizing human delta-1 and selete-1 can be prepared as shown in Example 9. In addition, genes expressed in cells using immunoglobulin genes isolated by various methods and gene cloning methods shown in the books (Antibodies a laboratory manual, E. Harlow et al., Cold Spring Harbor Laboratory) It can also be produced by recombinant antibodies. The antibody thus prepared can be used for purification of human delta-1 and delta-1. That is, by using these antibodies specifically recognizing human delta-1 and selete-1 shown in Example 9, it is possible to detect and measure human delta-1 and selete-1 of the present invention, and to differentiate cells. It can be used as a diagnostic agent for diseases associated with abnormalities such as malignant tumors.

  A more effective method as a purification method is affinity chromatography using an antibody. As the antibody used in this case, the antibody described in Example 9 can be used. In the case of a fusion protein, as shown in Example 8, an antibody against FLAG can be used for FLAG, and Protein G and Protein A can be used for human IgG Fc.

  As these fusion proteins, all fusion proteins other than the two examples shown here are possible. For example, histidine Tag, myc-tag, and the like can be mentioned. In addition to the known methods up to now, any fusion protein can be prepared using current genetic engineering techniques. The polypeptide of the present invention derived from is also included in the present invention.

  Intracellular signal transduction based on the physiological functions of human delta-1 and human serate-1 protein purified in this way based on various biological activity assays using biological individuals such as various cell lines, mice and rats, and molecular biological techniques. And various assay methods such as binding to a notch receptor.

  The present inventors have observed the effect on blood undifferentiated cells using IgG1 chimeric protein of human delta-1 and human selete-1.

  That is, as shown in Example 10, in the cord blood-derived blood undifferentiated cells enriched with the CD34 positive cell fraction, the polypeptide of the present invention has a colony-forming effect on blood undifferentiated cells that form colonies in the presence of various cytokines. It was found to have inhibitory activity. Moreover, this inhibitory activity was considered to have an unprecedented activity observed only in the presence of SCF.

  As shown in Example 11, human delta-1 or human serate-1 was subjected to long-term (8 weeks) liquid culture in the presence of various cytokines such as SCF, IL-3, IL-6, GM-CSF, and Epo. It was found that the addition of the IgG1 chimeric protein significantly prolonged the maintenance of colony forming cells. Furthermore, the effect | action which does not suppress the proliferation of colony forming cells was found. On the other hand, MIP-1α (Verfaillie et al., J. Exp. Med. 179, 643-649, 1994), a cytokine having blood cell migration and differentiation inhibitory action, has the effect of maintaining undifferentiation of blood undifferentiated cells. I didn't have it.

  Furthermore, as shown in Example 12, the most undifferentiated blood stem cell among human blood undifferentiated cells can be obtained by adding IgG1 chimeric protein of human delta-1 or human selete-1 to liquid culture in the presence of cytokines. It was found to have an activity that significantly maintains the number of LTC-IC (Long-Term Culture-Initiating Cells) that have been positioned.

  From these results, it is clear that human delta-1 and human selete-1 suppress the differentiation of blood undifferentiated cells, and their actions act from blood stem cells to colony forming cells. These physiological actions are necessary for the in vitro proliferation of blood undifferentiated cells. Particularly, cells cultured in a cell culture medium containing human delta-1 or human selete-1 are effective for recovery of bone marrow suppression after administration of an anticancer agent. Other conditions will allow for the expansion of hematopoietic stem cells in vitro. Furthermore, when used as a pharmaceutical, it has the effect of protecting and reducing the myelosuppressive action seen from side effects such as anticancer agents.

  Furthermore, in the undifferentiated cells other than blood cells, the action of suppressing cell differentiation can be mainly expected, and the action of promoting tissue regeneration can be expected.

  For use as a pharmaceutical product, it is desirable that the polypeptide of the present invention be prepared in the form of a freeze-dried product together with an appropriate stabilizer such as human serum albumin and dissolved or suspended in distilled water for injection before use. . For example, it can be provided as an injection or infusion prepared at a concentration of 0.1 to 1000 μg / ml. The present inventors subdivided into vials so that the compound of the present invention was 1 mg / ml and human serum albumin 1 mg / ml, and the activity of the compound was retained over a long period of time. Further, when cells are cultured and activated in vitro, a lyophilized product or a solution can be prepared and added to a medium or immobilized on a container used for culture, as in the case of pharmaceuticals. Regarding the toxicity of the polypeptide of the present invention, 10 mg / Kg of any polypeptide was intraperitoneally administered to mice, but no mouse death was confirmed.

  In addition, the in vitro physiological activity of the present invention can be administered to any disease model mouse, or an animal such as a rat or monkey that exhibits symptoms similar to those of the disease, and the recovery of its physical and physiological functions. It becomes possible by examining abnormalities. For example, if the abnormality is related to hematopoietic cells, a myelopathy model mouse is prepared by administering a 5-FU anticancer agent, and the bone marrow cells and peripheral blood cells of the group not treated with the compound of the present invention are administered to this mouse. It becomes clear by examining the number and physiological function. Furthermore, when examining the culture and proliferation of hematopoietic undifferentiated cells containing hematopoietic stem cells outside the body, the mouse bone marrow cells are cultured using an incubator or the like, and the compound of the present invention is added at that time. In the group not added, the cells after culturing were transplanted into lethal dose-irradiated mice, and the degree of recovery of the result can be examined by using the survival rate, fluctuation of blood cell count, etc. as indicators. Of course, since these results can be extrapolated to humans, it is possible to obtain effective data as an evaluation of the efficacy of the present compound.

  When the compound of the present invention is used as a pharmaceutical, its indication is treatment of diseases associated with abnormal cell differentiation, such as leukemia and malignant tumors, and human-derived cells are cultured in vitro to maintain their original functions. Can be applied to cell therapy that allows cells to proliferate or have new functions, etc., or treatment that regenerates without damaging the functions originally possessed by the tissue when administered after regeneration after tissue damage. It is. The dose at that time depends on the form and the like, but specifically, it may be about 10 μg / Kg to about 10 mg / Kg.

  Moreover, it is desirable to express in a form capable of forming a multimer as a form having stronger physiological activity.

  As shown in Example 10, since the inhibitory activity of human delta-1 and human selete-1 is higher in the IgG chimeric protein having a dimer structure, a form capable of forming a multimer as a form having strong physiological activity It is desirable to express in

  Human delta-1 and human selete-1 having a multimeric structure are expressed as a chimeric protein with the Fc part of human IgG described in the Examples and expressed as a multimer having a disulfide bond formed by the hinge part of the antibody, The antibody recognition site is expressed as a chimeric protein that expresses at the C-terminus or N-terminus, and the expressed polypeptide containing the extracellular portion of the human serate reacts with an antibody that specifically recognizes the antibody recognition site at the C-terminus or N-terminus The method of forming a multimer by carrying out is mentioned. Furthermore, as another method, a fusion protein with only the hinge region part of an antibody is expressed to form a dimer by disulfide bond, or other effects on human delta-1 and human serate-1 activities are affected. A multimeric human delta having a high specific activity of a dimer or more composed of a fusion protein prepared so as to express a peptide in the form of generating a disulfide bond in a non-given manner at the C-terminal, N-terminal or other site 1 and human selete-1 can also be obtained. In addition, one or more polypeptides selected from polypeptides containing the amino acid sequence of SEQ ID NO: 2, 3, 5 or 6 in the sequence listing are genetically engineered in two or more in series or in parallel to express a multimeric structure. There is also a method to make it. In addition, any currently known method of giving a dimeric or higher multimeric structure can be applied. Therefore, the present invention also relates to a compound comprising a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, 3, 5 or 6 in the sequence listing having a dimer or higher form produced by genetic engineering techniques. Included in the invention.

  In addition, as another method, there is a method of multimerization using a chemical cross-linking agent. For example, dimethylsuberoimidate dihydrochloride that crosslinks lysine residues, N- (γ-maleimidobutyryloxy) succinimide that crosslinks with thiol groups of cysteine residues, and glutaraldehyde that crosslinks amino groups and amino groups A dimer or higher multimer can be formed using these cross-linking reactions. Accordingly, a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, 3, 5 or 6 in the sequence listing in the form of a dimer or higher multimer produced by a chemical cross-linking agent is included. The compounds are also included in the present invention.

  For adaptation to a medical method of proliferating and activating cells outside the body and returning the cells to the body, human delta-1 or human selete-1 having the above-mentioned form can be added directly to the medium, but it is immobilized. It is possible to do that as well. As an immobilization method, the polypeptide can be covalently bound to a culture vessel by using amino groups or carboxyl groups of these polypeptides, using an appropriate spacer, or using the above-mentioned crosslinking agent. . Therefore, a polypeptide containing the amino acid sequence of SEQ ID NO: 2, 3, 5 or 6 in the sequence listing having a form existing on the solid surface is also included in the present invention.

  In addition, since human delta-1 and human selete-1 existing in nature are cell membrane proteins, the differentiation-inhibiting action performed in the examples can also be achieved by co-culturing cells expressing these molecules and blood undifferentiated cells. Can be expressed. Therefore, a method for co-culturing a cell transformed with a DNA encoding the amino acid sequence of SEQ ID NO: 2 to 7 in the sequence listing and an undifferentiated cell is also included in the present invention.

  The cells to be expressed may be COS-7 cells shown in the Examples, but human-derived cells are desirable, and further cells to be expressed may be blood cells or somatic cells in the human body, not cell lines. Therefore, it can also be incorporated into gene therapy vectors and expressed in vivo.

  As shown in Example 10, the FLAG chimeric protein of human delta-1 or human selete-1, which is a low concentration monomer, does not suppress colony formation but conversely promotes colony formation. It is considered that this action expressed Notch receptor and Notch ligand molecule when blood undifferentiated cells divide, and the polypeptide of the present invention worked as an antagonist of the action. Therefore, the polypeptide having the amino acid sequence of SEQ ID NO: 1, 2, 4 or 5 in the sequence listing also has a colony formation promoting action by controlling its action concentration. Therefore, a polypeptide having the amino acid sequence of SEQ ID NO: 2, 3, 5 or 6 in the sequence listing having a colony formation promoting action is also included in the present invention.

  Further, from this fact, inhibiting the binding of the present invention molecule, that is, the polypeptide having the amino acid sequence of SEQ ID NO: 2 to 7 in the sequence listing and these receptors can be used as a means for finding a molecule or compound that promotes cell differentiation. it can. As the method, various methods such as a binding experiment using a radioisotope, a luciferase assay using a transcriptional regulatory factor group which is a downstream molecule of a Notch receptor, and a simulation on a computer by performing an X-ray structural analysis can be applied. Therefore, the present invention also includes a drug screening method using the polypeptides of SEQ ID NOs: 2 to 7 in the Sequence Listing.

  In addition, as shown in Example 13, a specific leukemia cell line can be fractionated by using an IgG chimeric protein of human delta-1 or human selete-1. This can therefore be used as a diagnostic agent for leukemia and can be applied to the separation of specific blood cells. This result is considered to be because human delta-1 and human serate-1 molecule specifically bind to the receptor Notch receptor molecule. For example, when the above-mentioned extracellular portion and human IgG Fc fusion protein are used, the Notch receptor Expression can be detected. Notch is known to be associated with certain types of leukemia (Ellisen et al., Cell 66, 649-661, 1991), and therefore the amino acid sequences of SEQ ID NOs: 2, 3, 5 and 6 in the sequence listing Can be used as an in vitro or in vivo diagnostic agent.

  Examples of embodiments for carrying out the invention will be shown below, but the present invention is not necessarily limited thereto.

Example 1 Cloning of PCR product with human delta-1 primer and determination of nucleotide sequence Mixed primer corresponding to the amino acid sequence conserved in C-delta-1 and X-delta-1, ie, sense primer DLTS1 (SEQ ID NO: in the sequence listing) 11) and the antisense primer DLTA2 (described in SEQ ID NO: 12).

  Synthetic oligonucleotides were prepared using a fully automatic DNA synthesizer based on the solid phase method. As a fully automatic DNA synthesizer, 391 PCR-MATE, Applied Biosystems, USA was used. Nucleotides, 3′-nucleotide immobilized carriers, solutions and reagents were used according to the company's instructions. The predetermined coupling reaction was completed, and the oligonucleotide carrier from which the 5'-terminal protecting group had been removed with trichloroacetic acid was left in concentrated ammonia for 1 hour at room temperature to release the oligonucleotide from the carrier. Next, in order to release the protecting group for nucleic acid and phosphate, the reaction solution containing the nucleic acid was left in a sealed vial at 55 ° C. for 14 hours or more in a concentrated ammonia solution. Purification of each oligonucleotide liberated carrier and protecting group was performed using an Applied Biosystems OPC cartridge and detritylated with 2% trifluoroacetic acid. The purified primer was dissolved in deionized water so as to have a final concentration of 100 pmol / μl and used for PCR.

Amplification by PCR using these primers was performed as follows. Using 1 μl of human fetal brain-derived cDNA mixed solution (QUICK-Clone cDNA, CLONTECH, USA), 10 × buffer (500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mM MgCl ₂ , 0.01% gelatin) 5 μl, dNTP Mixture (manufactured by Japan Takara Shuzo Co., Ltd.) 4 μl, sense primer DLTS1 (100 pmol / μl) specific to the above vertebrate delta homolog and 5 μl of antisense primer DLTA2 (100 pmol / μl), and Taq DNA polymerase (AmpliTaq: (Nippon Kokuho Shuzo Co., Ltd., 5 U / μl) 0.2 μl is added. Finally, deionized water is added to make the total volume 50 μl. The process consists of 95 ° C. for 45 seconds, 42 ° C. for 45 seconds, and 72 ° C. for 2 minutes. This line as one cycle The cycle consisting of 95 ° C for 45 seconds, 50 ° C for 45 seconds, and 72 ° C for 2 minutes is defined as one cycle. This cycle is performed for 35 cycles, and finally the sample is left at 72 ° C for 7 minutes to perform PCR. went. A portion of this PCR product was subjected to 2% agarose gel electrophoresis, stained with ethidium bromide (Nihon Gene, Japan), and observed under ultraviolet light to confirm that about 400 bp cDNA was amplified. .

  The total amount of the PCR product was electrophoresed on a 2% agarose gel prepared with low melting point agarose (manufactured by GIBCO BRL, USA), stained with ethidium bromide, and after UV irradiation, about 400 bp of the PCR product by the delta primer. Cut out the band, add distilled water of the same volume as the gel, heat at 65 ° C. for 10 minutes to completely dissolve the gel, add an equal amount of TE saturated phenol (manufactured by Nippon Gene Co., Ltd. in Japan), 15000 rpm 5 After centrifuging for 5 minutes, the supernatant was separated, and a similar separation operation was carried out with TE saturated phenol: chloroform (1: 1) solution and further chloroform. DNA was recovered from the finally obtained solution by ethanol precipitation.

  PCRII Vector (manufactured by Invitrogen, USA, hereinafter referred to as pCRII) was used as a vector, mixed so that the molar ratio of the vector and the previous DNA was 1: 3, and the vector was used with T4 DNA ligase (manufactured by Invitrogen, USA). DNA was incorporated into the. PCRII into which DNA is incorporated is introduced into Escherichia coli One Shot Competent Cells (manufactured by Invitrogen, USA), and L-Broth (manufactured by Sigma, USA) semi-solid medium containing 50 μg / ml of ampicillin (manufactured by Sigma, USA) Seed on a plate and left at 37 ° C. for about 12 hours. The appearing colonies were randomly selected, planted in 2 ml of L-Broth liquid medium containing the same concentration of ampicillin, and cultured with shaking at 37 ° C. for about 18 hours. The plasmid is separated using a wizard miniprep (manufactured by Promega, USA) according to the attached instructions, and this plasmid is digested with the restriction enzyme EcoRI to cleave about 400 bp of DNA. Confirm that the PCR product is integrated, and confirm the clone The base sequence of the incorporated DNA was determined with a fluorescent DNA sequencer (Model 373S) manufactured by Applied Biosystems, USA.

Example 2 Full-Length Cloning and Disruption of a Novel Human Delta-1 Gene A clone having a full-length cDNA by plaque hybridization from a human placenta-derived cDNA library (cDNA inserted into λgt-11, manufactured by CLONTECH, USA) The search was performed from plaques corresponding to 1 × 10 ⁶ . The appearing plaque was transferred to a nylon filter (Hybond N +: manufactured by Amersham, USA), and the transferred nylon filter was treated with alkali (left on filter paper soaked with 1.5 M NaCl and 0.5 M NaOH for 7 minutes), then Neutralization treatment (1.5 M NaCl, 0.5 M Tris-HCl (pH 7.2), left on a filter paper soaked with 1 mM EDTA for 3 minutes) was performed twice, and then an SSPE solution (0.36 M NaCl, 0 .02M sodium phosphate (pH 7.7), 2 mM EDTA) in 2 times solution, shaken for 5 minutes, washed, and air dried. Then, it was left on a filter paper soaked with 0.4 M NaOH for 20 minutes, washed with 5 times concentrated SSPE solution for 5 minutes, washed and air-dried again. Using this filter, screening was performed with a human delta-1 probe labeled with radioisotope ³² P.

The DNA probe prepared in Example 1 labeled with the radioactive isotope ³² P was prepared as follows. Specifically, a pCRII into which a purified PCR product (about 400 bp) using a human delta-1 primer whose gene sequence was determined was cut out from a vector with EcoRI, and a DNA fragment was purified and recovered from a low melting point agarose gel. The obtained DNA fragment was labeled using a DNA labeling kit (Megaprime DNA labeling system: manufactured by Amersham, USA). Specifically, 5 μl of primer solution and deionized water were added to 25 ng of DNA to make a total volume of 33 μl, followed by boiling water bath for 5 minutes. Thereafter, 10 μl of reaction buffer containing dNTP, 5 μl of α- ³² P-dCTP, and 2 μl of T4 DNA polynucleotide kinase solution were added. In addition, it was bathed at 37 ° C. for 10 minutes, and then purified with a Sephadex column (Quick Spin Column Sephadex G-50: manufactured by Boehringer Mannheim, Germany), followed by boiling in a boiling water bath for 5 minutes and then ice-cooling for 2 minutes. used.

A filter prepared by the above-described method was prepared by adding a SSPE solution having a final concentration of 5 times each component, a Denhardt solution having a concentration of 5 times (manufactured by Japan Wako Pure Chemical Industries, Ltd.), 0.5% SDS (sodium dodecyl sulfate, After soaking in a prehybridization solution, which is a salmon sperm DNA (manufactured by Sigma, USA) denatured in a boiling water bath of 10 μg / ml, and shaken at 65 ° C. for 2 hours, In this method, the sample was immersed in a hybridization solution having the same composition as the prehybridization solution containing the ³² P-labeled probe, and shaken at 55 ° C. for 16 hours to perform hybridization.

  Next, the filter is immersed in an SSPE solution containing 0.1% SDS, shaken at 55 ° C., washed twice, and further immersed in an SSPE solution diluted 10-fold containing 0.1% SDS, and 4 ° C. at 55 ° C. Washed twice. The filter after washing was subjected to autoradiography using an intensifying screen. As a result, a clone of the strongly exposed portion was picked up, the plaque was re-wound and screened by the above-mentioned method, and a single clone was completely separated.

The isolated phage clone was 7 clones. According to the method of the written book, about 1 × 10 ⁹ pfu of phages of all these clones are prepared, phage DNA is purified, digested with restriction enzyme EcoRI, and similarly digested with EcoRI (produced by Stratagene, USA) Incorporated. When the DNA sequences at both ends of these clones were analyzed with a DNA sequencer, the three clones D5, D6, and D7 were all clones containing the 1st to 2244th sequences of the DNA sequence of SEQ ID NO: 8 in the sequence listing, and D4 These clones were clones containing 999 to 2663 of the DNA sequence of SEQ ID NO: 8 in the Sequence Listing. For the clones D5 and D4, a deletion mutant was prepared using a kilo-sequence deletion kit (manufactured by Nippon Treasure Shuzo Co., Ltd.) according to the attached instructions, and both 5 ′ and 3 ′ directions were prepared using the DNA sequencer. From the above, the full-length cDNA base sequence of the present invention was determined.

  Further, using the XhoI site at position 1214 of the DNA sequence of SEQ ID NO: 8 in the sequence listing, D4 and D5 were digested with the restriction enzyme XhoI, and plasmid pBSDe1-1 containing the full length DNA sequence of SEQ ID NO: 7 in the sequence listing was obtained. Produced.

Example 3 Cloning of Human Serate-1 Specific PCR Product and Determination of Base Sequence Mixed primer corresponding to the amino acid sequence conserved in Drosophila selete and rat jagd, ie, sense primer SRTS1 (described in SEQ ID NO: 13 in the sequence listing) and Antisense primer SRTA2 (described in SEQ ID NO: 14 in the sequence listing) was used. The manufacturing method was performed by the method described in Example 1.

  Amplification by PCR using these primers was performed as follows. Using 1 μl of the human fetal brain-derived cDNA mixed solution, 5 μl of 10 × buffer solution (described in Example 1), 4 μl of the dNTP Mixture, 5 μl of the sense primer SRTS1 (100 pmol / μl) specific for the above-mentioned Serate-1 homolog and Add 5 μl of the antisense primer SRTA2 (100 pmol / μl) and 0.2 μl of the Taq DNA polymerase, and finally add deionized water to make a total volume of 50 μl to 95 ° C. for 45 seconds, 42 ° C. for 45 seconds, 72 ° C. for 2 The process consisting of minutes is defined as one cycle, and this process is performed for 5 cycles. Furthermore, the process consisting of 95 ° C for 45 seconds, 50 ° C for 45 seconds, and 72 ° C for 2 minutes is performed as one cycle, and this process is performed for 35 cycles. PCR was carried out by standing at 72 ° C. for 7 minutes. A portion of this PCR product was subjected to 2% agarose gel electrophoresis, stained with ethidium bromide, and observed under ultraviolet light to confirm that about 500 bp of cDNA was amplified.

  The entire amount of the PCR product was electrophoresed on a 2% agarose gel prepared with the low melting point agarose, stained with ethidium bromide, and a band of about 500 bp was cut out under ultraviolet irradiation, and distilled water having the same volume as the gel was used. In addition, after heating at 65 ° C. for 10 minutes to completely dissolve the gel, an equal amount of TE saturated phenol is added, the supernatant is separated after centrifugation at 15,000 rpm for 5 minutes, and a similar separation operation is performed with TE saturated phenol: The reaction was carried out in a chloroform (1: 1) solution and further in chloroform. DNA was recovered from the finally obtained solution by ethanol precipitation.

  Using the pCRII Vector as a vector, the vector and the previous DNA were mixed so that the molar ratio was 1: 3, the DNA fragment was incorporated into the vector pCRII as in Example 1, and the gene was similarly introduced into Escherichia coli. The colonies that emerged were randomly selected, planted in 2 ml of L-Broth liquid medium containing the same concentration of ampicillin, cultured at 37 ° C. for about 18 hours with shaking, and the cells were collected and attached using the wizard miniprep. The plasmid was isolated according to the instructions in (2) and digested with the restriction enzyme EcoRI to confirm that the PCR product had been incorporated by cutting out about 500 bp of DNA. The base sequence of the incorporated DNA was determined with the DNA sequencer.

Example 4 Full-Length Cloning and Analysis of Novel Human Serate-1 Gene A clone having a full-length cDNA was searched from plaques corresponding to 1 × 10 ⁶ by plaque hybridization from the aforementioned human placenta-derived cDNA library. A filter was prepared by the method described in Example 2, and screening was performed using this filter with a human serate-1 probe labeled with a radioactive isotope ³² P.

The previous DNA probe labeled with the radioactive isotope ³² P was prepared according to the method described in Example 2, and hybridization, filter washing, and clone separation were performed according to the method described in Example 2.

Isolated phage clones were 22 clones. According to the written method, phages of all these clones were prepared at about 1 × 10 ⁹ pfu, and the phage DNA was purified, digested with restriction enzyme EcoR1, and incorporated into pBluescript similarly digested with EcoR1. When the DNA sequences at both ends of these clones were analyzed with a DNA sequencer, both clones S16 and S20 were clones containing the 1st to 1873th sequences of the DNA sequence of SEQ ID NO: 9 in the sequence listing, and S5 and S14 These 2 clones were clones containing DNA sequences 990 to 4005 of SEQ ID NO: 9 in the Sequence Listing. These clones produced a deletion mutant according to the attached instructions using the kilosequence deletion kit, and encoded the polypeptide of the present invention from both the 5 ′ direction and the 3 ′ direction using the DNA sequencer. The cDNA base sequence to be determined was determined.

  Further, using the BglII site at position 1293 of the DNA sequence of SEQ ID NO: 9 in the sequence listing, S20 and S5 were digested with the restriction enzyme BglII, and the gene sequences from 1 to 4005 of the gene sequence of SEQ ID NO: 9 in the sequence listing were The DNA is subcloned into the E. coli vector pBluescript, and this plasmid is designated as pBSSRT.

As a result, it was found that a stop codon was not observed at the C-terminal, and the region encoding the C-terminal amino acid sequence of the intracellular portion could not be cloned. Therefore, the full-length gene was cloned from GIBCO-BRL, USA 3 Using the 'RACE system kit, the cDNA of the 3'-directional gene was cloned from human placenta-derived polyA ⁺ RNA (manufactured by CLONTECH, USA), and the gene sequence was determined.

  Using the restriction enzyme Bgl2 site at position 1293 and the Acc1 site at position 3943 of the DNA sequence of SEQ ID NO: 9 in the sequence listing, the three gene fragments thus gene cloned were used to obtain the full length DNA sequence of SEQ ID NO: 5 in the sequence listing. Was ligated between EcoR1 and Xba1 of the multi-cloning site of pUC18 to prepare pUCSR-1 containing the full-length gene of human serate-1. The sequence of this gene is shown in SEQ ID NO: 9 in the sequence listing together with the amino acid sequence.

Example 5 Preparation of Human Delta-1 Expression Vector Using the gene consisting of the DNA sequence shown in SEQ ID NO: 7 in the Sequence Listing, the following expression vectors for human delta-1 protein listed in 1) to 5) were prepared. The addition of restriction enzyme sites and the insertion of a short gene sequence were all carried out according to the attached instruction manual using ExSite PCR-Based Site-Directed Mutagenesis Kit manufactured by Stratagene.

1) Secreted human delta-1 protein (HDEX) expression vector A cDNA encoding the polypeptide of No. 1 to No. 520 of the amino acid sequence of SEQ ID No. 3 in the sequence listing is linked to the expression vector pMKITNeo containing SRα promoter and neomycin resistance gene. An expression vector was prepared.

  In preparing the expression vector of human delta-1, in order to express the gene product more stably, EcoRI is located in the upstream portion of 20 bp in the 5 ′ direction of the start codon (the number 179 of the gene sequence of SEQ ID NO: 8 in the sequence listing). Added a site. That is, using the above Mutagenesis Kit, the DNA sequence described in SEQ ID NO: 8 in the sequence listing, and the plasmid pBSDel-1 containing the full-length cDNA of human delta-1 as a template, the genes of SEQ ID NO: 15 and SEQ ID NO: 16 in the sequence listing Using an oligonucleotide having a sequence as a primer, a DNA having an EcoRI site added to the portion 20 bp upstream in the 5 ′ direction was prepared. This plasmid is hereinafter referred to as pBS / Eco-Delta.

  Next, using this pBS / Eco-Delta as a template, a termination codon and further a restriction enzyme MluI site are added to the carboxyl terminal portion. Similarly, using Mutagenesis Kit, the gene sequences of SEQ ID NO: 17 and SEQ ID NO: 18 in the sequence listing are used. A stop codon and further an MluI site were added using the oligonucleotide having an oligonucleotide as a primer. Next, this vector was digested with EcoRI and MluI, and the gene fragment of about 1600 bp cut out was connected to pMKITNeo treated with the same restriction enzyme to construct an expression vector. This vector was named pHDEX.

2) Expression vector of secreted human delta-1 FLAG chimeric protein (HDEXFLAG) encoding a chimeric protein in which a cDNA encoding a FLAG sequence is added to the C-terminal of the polypeptide of amino acids 1 to 520 of SEQ ID NO: 3 in the sequence listing The cDNA to be ligated was ligated to an expression vector pMKITNeo containing an SRα promoter and a neomycin resistance gene to prepare an expression vector.

  Using pBS / Eco-Delta as a template, add a FLAG sequence after the carboxy terminal portion of the extracellular portion, that is, the 520th Gly of SEQ ID NO: 3 in the sequence listing, and then add a stop codon and then a restriction enzyme MluI site. Therefore, similarly, using Mutagenesis Kit, using the oligonucleotides having the gene sequences of SEQ ID NO: 19 and SEQ ID NO: 18 in the sequence listing as primers, adding a gene encoding a FLAG sequence at the C-terminus, a stop codon, and further an MluI site It was. Next, this vector was digested with EcoRI and MluI, and the expression vector was constructed by connecting the excised gene fragment of about 1700 bp to pMKITNeo treated with the same restriction enzyme. This vector was named pHDEXFLAG.

3) Expression vector of secreted human delta-1 IgG1 Fc chimeric protein (HDEXIg) Poly having the amino acid sequence of SEQ ID NO: 3 in the sequence listing added the amino acid sequence of the Fc part below the hinge part of human IgG1 to the C-terminal of the polypeptide The gene sequence encoding the peptide was connected to pMKITNeo to prepare an expression vector.

  Preparation of a fusion protein with an immunoglobulin Fc protein uses a gene using genomic DNA containing an intron according to the method of Zettlmeissl et al. (Zettlmeissl et al., DNA cell Biol., 9, 347-354, 1990) The gene was produced using the PCR method. That is, using human genomic DNA as a template, the gene sequence encoding the human IgG1 Fc portion is an oligonucleotide having the sequence of SEQ ID NO: 20 in the sequence listing with the restriction enzyme BamHI site, and the sequence listing with the restriction enzyme XbaI site. PCR was performed using the oligonucleotide having the sequence of SEQ ID NO: 21 as a primer, a band of about 1.4 kbp was purified, treated with restriction enzymes BamHI and XbaI (manufactured by Takara Shuzo Co., Ltd.), and the same restriction enzyme The treated pBluescript was ligated with T4 DNA ligase and subcloned. Thereafter, this plasmid DNA was purified and sequenced to confirm the gene sequence, and it was confirmed that the gene sequence was indeed the genomic DNA corresponding to the hinge part of the heavy chain of human IgG1 (the sequence was determined by Kabat et al. , Sequence of Immunological Interest, NIH publication No 91-3242, 1991). Hereinafter, this plasmid is referred to as pBShIgFc.

  Next, using the pBS / Eco-Delta as a template, similarly, using Mutagenesis Kit, a restriction enzyme BamHI site was added after the carboxyl terminal portion of the extracellular portion, that is, the 520th Gly of SEQ ID NO: 3 in the Sequence Listing. Further, in order to add restriction enzyme XbaI and MluI sites downstream thereof, addition of BamHI, XbaI, and MluI sites using the Mutagenesis Kit in the same manner as the oligonucleotides of SEQ ID NO: 22 and SEQ ID NO: 23 in the sequence listing. Went. This vector is digested with XbaI and BamHI, and the above-mentioned pBShIgFc is digested with XbaI and BamHI to connect the gene fragment of about 1200 bp, which finally encodes the target secreted human delta-1 IgG1Fc chimeric protein. A vector containing the gene fragment to be prepared was prepared. Finally, this vector was digested with EcoRI and MluI, and an expression vector was constructed by connecting the excised gene fragment of about 3000 bp to pMKITNeo treated with the same restriction enzyme. This vector was named pHDEXIg.

4) Full-length human delta-1 protein (HDF) expression vector A cDNA encoding the polypeptide from No. 1 to No. 702 of the amino acid sequence of SEQ ID NO: 4 in the sequence listing is transferred to the expression vector pMKITNeo containing the SRα promoter and the neomycin resistance gene. The expression vector was produced by ligation.

  Using pBS / Eco-Delta as a template, similarly using Mutagenesis Kit to add a stop codon and a restriction enzyme MluI site after the 702th Val of SEQ ID NO: 4 in the sequence listing Using oligonucleotides having the gene sequences of SEQ ID NO: 24 and SEQ ID NO: 25 in the column table as primers, a stop codon and further an MluI site were added to the C-terminus. Next, this vector was digested with EcoRI and MluI, and an about 2200 bp gene fragment excised was connected to pMKITNeo treated with the same restriction enzyme to construct an expression vector. This vector was named pHDF.

5) FLAG chimeric protein (HDFLAG) expression vector of full-length human delta-1 encoding a chimeric protein in which a cDNA encoding the FLAG sequence is added to the C-terminal of the polypeptide of amino acids 1 to 702 of SEQ ID NO: 4 in the sequence listing The cDNA to be ligated was ligated to an expression vector pMKITNeo containing an SRα promoter and a neomycin resistance gene to prepare an expression vector.

  Using pBS / Eco-Delta as a template, a FLAG sequence is added to the carboxyl terminal portion, followed by a stop codon and a restriction enzyme MluI site. Using an oligonucleotide as a primer, a gene encoding a FLAG sequence at the C-terminus, a stop codon, and an MluI site were added.

  From this vector, DNA encoding the full length of human delta-1 was cloned into E. coli vector pUC19 to prepare vector pUCDL-1F encoding full length human delta-1.

  Next, this vector was digested with EcoRI and MluI, and an about 2200 bp gene fragment excised was connected to pMKITNeo treated with the same restriction enzyme to construct an expression vector. This vector was named pHDFLAG.

Example 6 Preparation of New Human Serite-1 Expression Vector Using the gene consisting of the DNA sequence shown in SEQ ID NO: 9 in the Sequence Listing, the following 6) and 10) human serate-1 protein expression vectors were prepared. The addition of restriction enzyme sites and the insertion of short gene sequences were all performed using ExSite PCR-Based Site-Directed Mutagenesis Kit according to the attached instruction manual.

6) Secreted human Serreito 1 (HSEX) expression vector A cDNA encoding a chimeric protein added with a cDNA encoding the polypeptide of No. 1 to No. 1036 of the amino acid sequence of SEQ ID No. 6 in the sequence listing was linked to the expression vector pMKITNeo, An expression vector was prepared.

  In producing an expression vector of a polypeptide-expressing cell having the amino acid sequence from 1 to 1036 of the amino acid sequence of SEQ ID NO: 6 in the sequence listing, in order to express the gene product more stably, a start codon (SEQ ID NO: An EcoRI site was added to the 10 bp upstream portion in the 5 ′ direction of No. 409 of 9 gene sequences). That is, using the above-mentioned Mutagenesis Kit, a plasmid pBSSRT containing the human selate-1 cDNA of No. 1 to No. 4005 of the DNA sequence shown in SEQ ID No. 9 of the Sequence Listing as a template, and an oligo having the gene sequence of SEQ ID No. 27 of the Sequence Listing Using an oligonucleotide having the nucleotide and the gene sequence of SEQ ID NO: 28 in the Sequence Listing as a primer, DNA having an EcoRI site added to the 10 bp upstream portion in the 5 ′ direction was prepared.

  Next, using the thus prepared vector (hereinafter referred to as pBS / Eco-Serrate-1) as a template, it terminates at the carboxyl terminal portion of the extracellular portion, that is, the carboxyl terminal of the polypeptide of SEQ ID NO: 6 in the sequence listing. In order to add a codon and further a restriction enzyme MluI site, similarly, using Mutagenesis Kit, using an oligonucleotide having the gene sequence of SEQ ID NO: 29 of the sequence listing and an oligonucleotide having the gene sequence of SEQ ID NO: 30 of the sequence listing as primers, A stop codon and an MluI site were added. Next, this vector was digested with EcoRI and MluI, and an expression vector was constructed by connecting the excised gene fragment of about 3200 bp to pMKITNeo treated with the same restriction enzyme. This vector was named pHSEX.

7) Expression vector of FLAG chimeric protein (HSEXFLAG) of split human selate-1 cDNA encoding a FLAG chimeric protein having a FLAG sequence at the C-terminal of the polypeptide of amino acids 1 to 1036 of SEQ ID NO: 6 in the sequence listing The expression vector was constructed by connecting to the expression vector pMKITNeo containing the SRα promoter and the neomycin resistance gene.

  Using pBS / Eco-Serrate-1 as a template, add a FLAG sequence to the carboxyl terminal part of the extracellular portion, that is, the carboxyl terminal of the polypeptide of SEQ ID NO: 6 in the sequence listing, and then add a stop codon and further a restriction enzyme MluI site. Similarly, using Mutagenesis Kit, using the oligonucleotide having the gene sequence of SEQ ID NO: 31 of the Sequence Listing and the oligonucleotide having the gene sequence of SEQ ID NO: 30 of the Sequence Listing as primers, a gene encoding a FLAG sequence and a stop codon And an MluI site was added. Next, this vector was digested with EcoRI and MluI, and an expression vector was constructed by connecting the excised gene fragment of about 3200 bp to pMKITNeo treated with the same restriction enzyme. This vector was named pHSEXFLAG.

8) Expression vector of secreted human selete-1 IgG1Fc chimeric protein (HSEXIg) The amino acid sequence of the Fc part below the hinge part of human IgG1 was added to the C-terminal of the polypeptide having the amino acid sequence shown in SEQ ID NO: 6 in the Sequence Listing. The gene sequence encoding the polypeptide was connected to pMKITNeo to prepare an expression vector.

  Using pBS / Eco-Serrate as a template, similarly using Mutagenesis Kit, a restriction enzyme BamHI site was added after the polypeptide having the carboxyl terminal portion of the extracellular portion, that is, the sequence of SEQ ID NO: 6 in the sequence listing, Furthermore, in order to add restriction enzyme XbaI and MluI sites downstream thereof, using an oligonucleotide having the gene sequence of SEQ ID NO: 32 of the sequence listing and an oligonucleotide having the sequence of SEQ ID NO: 33 of the sequence listing as primers, BamHI, XbaI and MluI sites were added. This vector is digested with XbaI and BamHI, and the above-mentioned pBShIgFc is digested with XbaI and BamHI, and then the gene fragment of about 1200 bp that is excised is connected to finally encode the IgG1Fc chimeric protein of the target secreted human serate-1 A vector containing the gene fragment to be prepared was prepared. Finally, this vector was digested with EcoRI and MluI, and an expression vector was constructed by connecting the excised gene fragment of about 4400 bp to pMKITNeo treated with the same restriction enzyme. This vector was named pHSEXIg.

9) Protein (HSF) expression vector of full-length human serato 1 The cDNA encoding the polypeptide of amino acids 1 to 1187 of SEQ ID NO: 7 in the sequence listing is transferred to the expression vector pMKITNeo containing SRα promoter and neomycin resistance gene. The expression vector was produced by ligation.

  For the production of a full-length expression vector, pBS / Eco-Serrate-1 is digested with restriction enzymes EcoRI and BglII, and the excised gene fragment of about 900 bp is digested with pUCSR-1 with the same restriction enzyme, and then ligated. Thus, a vector pUC / Eco-Serrate-1 encoding the human selate-1 full-length gene was prepared.

  Using this pUC / Eco-Sererate-1 as a template, in order to add a stop codon and a restriction enzyme MluI site after the 1187th Val of SEQ ID NO: 7 in the sequence listing, a Mutagenesis Kit is similarly used. Using the oligonucleotides having the gene sequences of SEQ ID NO: 34 and SEQ ID NO: 35 in the sequence listing as primers, a stop codon and further an MluI site were added to the C-terminus. Next, this vector was digested with EcoRI and MluI, and an expression vector was constructed by connecting the excised gene fragment of about 3700 bp to pMKITNeo treated with the same restriction enzyme. This vector was named pHSF.

10) FLAG chimeric protein (HSFLAG) expression vector of full-length human selete-1 A chimeric protein in which a cDNA encoding a FLAG sequence is added to the C-terminal of the polypeptide of amino acids 1 to 1187 of SEQ ID NO: 7 in the sequence listing The encoding cDNA was ligated to the expression vector pMKITNeo containing the SRα promoter and the neomycin resistance gene to prepare an expression vector.

  Using pUC / Eco-Serrate-1 as a template, add a FLAG sequence to the carboxyl terminal portion, then add a stop codon and a restriction enzyme MluI site in the same manner as shown in SEQ ID NO: 36 and SEQ ID NO: 35 in the sequence listing. Using the oligonucleotide as a primer, a gene encoding a FLAG sequence at the C-terminus, a stop codon, and an MluI site were added. Next, this vector was digested with EcoRI and MluI, and an expression vector was constructed by connecting the excised gene fragment of about 3700 bp to pMKITNeo treated with the same restriction enzyme. This vector was named pHSFLAG.

Example 7 Gene Introduction and Expression of Various Expression Vectors into Cells The expression vectors prepared in Examples 5 and 6 were introduced into COS-7 cells (available from RIKEN, Cell Development Bank, RCB0539).

The cells before gene introduction were cultured in D-MEM (Dulbecco's modified MEM medium, manufactured by GIBCO-BRL, USA) 10% FCS. The cell culture medium was changed on the day before gene transfer, and the cells were cultured overnight at 5 × 10 ⁵ cells / ml. On the day of gene introduction, the cells are precipitated by centrifugation, washed twice by centrifugation with PBS (−), and then prepared to 1 × 10 ⁷ cells / ml in 1 mM MgCl ₂ and PBS (−). did. Gene transfer was performed by electroporation using a gene transfer device Gene Pulser manufactured by Bio-Rad, USA. The above cell suspension was placed in a 500 μl electroporation dedicated cell (0.4 cm), 20 μg of the expression vector was added, and the mixture was left on ice for 5 minutes. Thereafter, voltage was applied twice under the conditions of 3 μF and 450 V, and the two times were left at room temperature for 1 minute. Thereafter, the cells were allowed to stand in ice for 5 minutes, and then the cells were seeded on a 10 cm diameter cell culture dish in which 10 ml of the above medium had been dispensed in advance, and cultured in a 37 ° C., 5% carbon dioxide incubator.

  The next day, the culture supernatant is removed, and the cells attached to the dish are washed twice with 10 ml of PBS (−). In the case of the expression vectors pHDEX, pHDEXFLAG, pHDEXIg, pHSEX, pHSEXFLAG, and pHSEXIg, 10 ml of serum-free D-MEM The culture supernatant was recovered, and the buffer was replaced with PBS (-) with Centricon 30 (Amicon, USA), and at the same time 10-fold concentrated to obtain a cell culture supernatant.

In the case of pHDF, pHDFLAG, pHSF, and pHSFLAG, the medium was replaced with D-MEM containing 10% FCS, and the cells were further cultured for 3 days to prepare cell disruptions. That is, 2 × 10 ⁶ cells were suspended in 200 μl of cell lysis buffer (50 mM Hepes (pH 7.5), 1% TritonX100, 10% glycerol, 4 mM EDTA, 50 μg / ml Aprotinin, 100 μM Leupeptin, 25 μM Pepstatin A, 1 mM PMSF). It became cloudy, left in ice for 20 minutes, and then centrifuged at 14,000 rpm for 20 minutes to remove the supernatant and obtain a cell disruption.

  Using the sample thus obtained, protein expression was confirmed by Western blotting.

  That is, using the SDS-PAGE electrophoresis tank and SDS-PAGE polyacrylamide gel (gradient gel 5-15%) manufactured by ACI Japan, the concentrated culture supernatant or cell disrupted product, the attached instruction manual SDS-PAGE was performed according to Samples were 2-mercaptoethanol (2-ME) added and reduced by boiling water bath treatment for 5 minutes, and non-reduced samples not subjected to this treatment, and the marker was Amersham's Rainbow A marker (for high molecular weight) was used, and a sample buffer and a migration buffer were prepared according to the attached instruction manual. After completion of SDS-PAGE, the acrylamide gel was transferred to a PVDF membrane filter (manufactured by BioRad, USA) using a mini-transblot cell manufactured by the same company.

  The filter thus prepared was shaken at 4 ° C. overnight in Block Ace (manufactured by Nippon Nippon Pharmaceutical Co., Ltd.), TBS-T (20 mM Tris, 137 mM NaCl (pH 7.6), 0.1% Tween 20). Blocked. According to the instructions attached to the ECL Western blotting detection system (Amersham, USA), when the target protein is derived from human delta-1, the anti-human delta-1 mouse monoclonal antibody described in Example 9 was used as the primary antibody, Anti-human selete-1 mouse monoclonal antibody described in Example 9 is used as the primary antibody when derived from -1, and mouse monoclonal antibody Anti-FLAG M2 (manufactured by Kodak, USA) is used as the primary antibody in the case of FLAG chimera. Then, a peroxidase-labeled anti-mouse Ig sheep antibody (Amersham, USA) was reacted as a secondary antibody. In the case of IgG chimera, a peroxidase-labeled anti-human Ig sheep antibody (Amersham, USA) was reacted.

  The reaction time of each antibody was allowed to react at room temperature for 1 hour, and the operation of shaking and washing with TBS-T for 10 minutes at room temperature was repeated three times between each reaction. After the final washing, the filter was immersed in a reaction solution of an ECL western blotting detection system (manufactured by Amersham, USA) for 5 minutes, wrapped in polyvinylidene chloride wrap and exposed to X-ray film.

  As a result, in the reduced sample, the protein obtained by introducing pHDEX and pHDEXFLAG was about 65 kDalton, the protein obtained by introducing pHDEXIg was about 95 kDalton, and the protein obtained by introducing pHDF and pHDFLAG was about A band of 85 kDalton was detected. On the other hand, in the non-reduced sample, when pHDEXIg is introduced, a slightly smeared band from 120 k to 200 k dalton mainly detects a band of about 180 k dalton, and has a molecular weight almost twice that of the reducing condition. It was confirmed that a monomer was formed.

  Similarly, in the reduced sample, the protein obtained by introducing pHSEX and pHSEXFLAG is about 140 kDalton, the protein obtained by introducing pHSEXIg is about 170 kDalton, and the protein obtained by introducing pHSF and pHSFLAG is A band of about 150 kDalton was detected. On the other hand, in the non-reduced sample, when pHSEXIg is introduced, a slightly smeared band of 250 k to 400 k dalton is mainly detected and a band of about 300 k dalton is detected, and the molecular weight is almost twice that of the reducing condition. It was confirmed that a monomer was formed.

  In these experiments, COS-7 cell lysate and culture supernatant into which pMKITNeo vector was introduced as a control were tested in the same manner. However, anti-human delta-1 mouse monoclonal antibody, anti-human selete-1 mouse monoclonal antibody, anti-FLAG antibody were tested. No band reacting with anti-human Ig antibody was detected.

  From the above results, all of these 10 expression vectors were able to produce the desired polypeptide.

Example 8 Purification of Secreted Human Delta-1 and Human Serite-1 Proteins Using Transfected Cells A large amount of COS-7 cell culture supernatant containing HDEXFLAG, HDEXIg, HSEXFLAG, and HSEXIg whose expression was detected by the method of Example 7 was prepared. Each chimeric protein was purified by an affinity column.

  For HDEXFLAG and HSEXFLAG, the 2 liter culture supernatant obtained by the method described in Example 7 was passed through a column packed with Anti-FLAG M2 Affinity Gel (manufactured by Kodak, USA), and the FLAG sequence possessed by the chimeric protein and The chimeric protein was adsorbed onto the column by the affinity of the gel Anti-FLAG antibody. As the column, a disposable column (manufactured by BioRad, USA) having an inner diameter of 10 mm was used, and 5 ml of the gel was packed. Adsorption was performed by assembling a circulating circuit of medium bottle → column → peristor pump → medium bottle and circulating for 72 hours at a flow rate of 1 ml / min. Thereafter, the column was washed with 35 ml of PBS (−) and eluted with 50 ml of 0.5 M Tris-glycine (pH 3.0). In advance, 200 μl of 0.5 M Tris-HCl (pH 9.5) is dispensed in a small tube (Falcon, USA 2063) in advance, and 25 ml each of the eluate is dispensed into the tube. Neutralized.

  10 μl of each of the elution fractions of the secreted FLAG chimeric protein purified by the above method was subjected to the reduction treatment described in Example 7, and subjected to SDS-PAGE electrophoresis using a 5-15% concentration gradient polyacrylamide gel. After completion of the electrophoresis, silver staining was performed using Wako Silver Dye Kit II manufactured by Wako Pure Chemical Industries, Ltd. according to the attached instructions. As a result, HSFLAG showed bands in the elution fractions from No. 4 to No. 8, and this molecular weight was consistent with the results of Western blotting using anti-FLAG antibody obtained in Example 6 and HDEXFLAG and HSEXFLAG. From these results, HDEXFLAG and HSEXFLAG pure products were purified.

  Regarding IgG1Fc chimeric proteins, ie, HDEXIg and HSEXIg, 2 liters of the culture supernatant was adsorbed on a Protein A Sepharose column manufactured by Pharmacia, Sweden, in the same manner as the FLAG chimeric protein, and the eluted fraction was fractionated.

  Similarly to the FLAG chimeric protein, a part of the eluate was used to determine the elution fraction, confirm the size, and test the purity by SDS-PAGE electrophoresis and silver staining under reducing conditions. As a result, bands were detected from No. 4 to No. 15 in the eluted fraction, and the size was consistent with the results of Western blotting using anti-human Ig antibody and HDEXIg and HSEXIg. From these results, HDEXIg and HSEXIg pure products were purified.

Example 9 Production of antibodies recognizing human delta-1 and human serate-1 Rabbits were immunized with HDEXFLAG and HSEXFLAG purified by the method described in Example 8, respectively, and after antibody titer measurement, whole blood was collected. The serum was collected, and the anti-human delta-1 rabbit polyclonal antibody and the anti-human seraito 1 rabbit polyclonal antibody were respectively purified using an Econopack serum IgG purification kit manufactured by BioRad, USA according to the attached instruction manual. did.

  A mouse monoclonal antibody was prepared according to the method described in the literature, using HDEXFLAG and HSEXFLAG purified by the method described in Example 8 as immunogens. Specifically, HDEXFLAG and HSEXFLAG purified as described above were separately immunized subcutaneously and intradermally in Balb / c mice (manufactured by Nippon SLC, Japan). After the second immunization, blood was collected from the fundus and the increase in the antibody titer in the serum was observed. After the third immunization, the mouse spleen cells were taken out, the mouse myeloma cell line P3X63Ag8 (ATCC TIB9) and the polyethylene glycol method Cell fusion. A hybridoma is selected in a HAT medium (manufactured by Japan Immunobiological Research Laboratories), and a hybridoma strain producing an antibody that recognizes the extracellular portion of human delta-1 or human serate-1 in the medium is isolated by an enzyme antibody method. A hybridoma-producing strain that produces a mouse monoclonal antibody that specifically recognizes human delta-1 or human selete-1 was established.

  The hybridoma culture supernatant thus established was prepared by purifying anti-human delta-1 monoclonal antibody and anti-human selete-1 monoclonal antibody using Mab TrapG II manufactured by Pharmacia, Sweden, according to the attached instruction manual. .

An affinity column was prepared using these monoclonal antibodies. The affinity column was prepared using CNBr activated Sepharose 4B manufactured by Pharmacia, Sweden, according to the attached instruction manual. A column having a size of ² cm ² × 1 cm was prepared from ² ml of this gel.

  The COS-7 cell culture supernatant concentrate into which pHDEX was introduced was introduced into the column to which the anti-human delta-1 monoclonal antibody was bound, and pHSEX was introduced into the column to which the anti-human selete-1 monoclonal antibody was bound. Each of the COS-7 cell culture supernatant concentrates was washed at a rate of 20 ml / hr, then washed with 15 ml of PBS (−) at the same rate, and finally washed with 0.1 M sodium acetate, 0.5 M NaCl (PH4. 0). 1 ml of this eluent was collected and neutralized by adding 200 μl of 1M Tris-HCl (pH 9.5) to each fraction.

  Furthermore, according to the method described in Example 8, each purified protein was subjected to SDS-PAGE under reducing conditions, silver staining and Western blotting were performed to estimate the molecular weight. As a result, about 65 kDalton HDEX was purified from the COS-7 cell culture supernatant concentrate into which pHDEX was introduced, and about 140 kDalton HDSEX was purified from the COS-7 cell culture supernatant concentrate into which pHSEX was introduced. It was confirmed that human delta-1 and human selete-1 can be purified using these affinity columns.

Example 10 Effect of HDEXIg and HSEXIg on colonization of blood undifferentiated cells To observe the physiological effect of HDEXIg and HSEXIg on blood undifferentiated cells, CD34 positive cells were treated with serum-free semisolid in the presence of HDEXIg or HSEXIg and existing cytokines. The cells were cultured in a medium, and the increase or decrease in colony forming cells was observed.

  For human umbilical cord blood or human normal bone marrow blood CD34 positive cells, umbilical cord blood or adult normal bone marrow blood is treated with silica solution (manufactured by Japan Immunobiological Research Institute) according to the attached instructions, and then Ficoll pack (Pharmacia, Sweden). The low density cell fraction (<1.077 g / ml) was separated from the fractionated mononuclear cells by a specific gravity centrifugation method.

  Separation of CD34 positive cells was performed according to the attached instruction manual using a microselector stem manufactured by AIS, USA or Dynabeads M-450 CD34 manufactured by Dynal, Norway, and DETAChaBEADS CD34. After separation, the purity is stained with FITC-labeled anti-CD34 antibody HPCA2 (manufactured by Becton Decktonson, USA) and tested with its flow cytometer (FACSCalibur) to confirm that it has a purity of 85% or higher. Used.

  400 CD34 positive cells thus separated are uniformly suspended so as to be present in 1 ml of the following medium, seeded in a 35 mm dish (Falcon, USA), 37 ° C., 5% carbon dioxide gas, 5% oxygen gas. After culturing for 2 weeks in a carbon dioxide incubator under an atmosphere of 90% nitrogen gas and 100% humidity, the formed blood cell colonies were counted under an inverted microscope.

The medium used for the culture was α-medium (manufactured by GIBCO-BRL, USA), 2% Deionized Bovine Serum Albumin (BSA, Sigma USA), 10 μg / ml human insulin (Sigma USA), 200 μg / ml transferrin (USA) Sigma), 10 ⁻⁵ M 2-mercaptoethanol (Nacalai Tesque, Japan), 160 μg / ml soybean lectin (Sigma, USA), 96 μg / ml cholesterol (Sigma, USA), 0.9% methylcellulose (Japan Wako Pure Chemical Industries, Ltd.)

  Human delta-1 extracellular Ig chimeric protein (HDEXIg) or human selete-1 extracellular Ig chimeric protein (final concentration of 1 μg / ml in the presence of cytokine under the following three conditions in the above medium ( HSEXIg) was added, and human IgGl (manufactured by Athens Research and Technology, USA) was added to the comparison group at the same concentration in order to see the effect of the IgGFc portion.

Cytokine conditions are as follows.
Condition 1: 100 ng / ml human SCF (Intergen, USA), 10 ng / ml human IL-3 (Intergen, USA), 100 ng / ml human IL-6 (Intergen, USA).
Condition 2: 100 ng / ml human SCF, 10 ng / ml human IL-3, 4 ng / ml human thrombopoietin (manufactured by Pepro Tech, USA).
Condition 3: 100 ng / ml human SCF, 10 ng / ml human IL-3, 100 ng / ml human IL-6, 2 U / ml Epo (manufactured by Chugai Pharmaceutical, Japan), 10 ng / ml human G-CSF (manufactured by Chugai Pharmaceutical, Japan) ).

  The results are shown in FIG. FIG. 2A shows the case of human delta-1 extracellular Ig chimeric protein (HDEXIg), and B shows the case of human selete-1 extracellular Ig chimeric protein (HSEXIg). A and B were human umbilical cord blood CD34-positive cells derived from different sources. The vertical axis represents the number of colonies, the white column is a comparison group, and the black column is data including HDEXIg or HSEXIg, respectively.

  Both HDEXIg and HSEXIg had an activity to suppress colony formation. There was no difference in the types of colony forming cells in these results. Therefore, the molecule of the present invention has a colony formation inhibitory effect on colony-forming cells of blood undifferentiated cells. That is, it became clear that it has a differentiation inhibitory action. When this activity was compared in the presence and absence of SCF, this inhibitory activity tended to be observed only in the presence of SCF.

  Further, the concentration dependence of this activity was examined. In addition, the dimer HSEXIg and the monomer HSEXFLAG were also compared. The results are shown in FIG. Although the concentration in this case is shown as a molar concentration, in order to compare the dimer and the monomer, the dimer HSEXIg is shown as twice the exact molar concentration, and the molar concentration of the human selete-1 moiety is Plots were made to be the same. The vertical axis is the number of colonies formed, and the horizontal axis is the molar concentration. The number of colonies formed without notch ligand was plotted on the vertical axis of 0 concentration. For comparison, the number of colonies formed of 1 μg / ml human IgG1 was about 100.

  From these results, although HSEXIg and HSEXFLAG inhibited colony formation in a concentration-dependent manner, the activity of HSEXIg, which is a dimer, was clearly stronger. Monomer HSEXFLAG was also observed to promote colony formation in the low concentration region.

Example 11 Effects of HDEXIg and HSEXIg on long-term liquid culture of colony-forming blood undifferentiated cells In order to observe the physiological effects of HDEXIg and HSEXIg on blood undifferentiated cells, cord blood CD34 positive cells were treated with HDEXIg or HSEXIg and existing cytokines. Then, liquid culture was carried out for a long time in a serum-free medium, and the increase or decrease in colony forming cells was observed.

Umbilical cord blood mononuclear cells CD34 positive cells separated by the method described in Example 10 were subjected to liquid culture at 1000 cells / well in a 24-well cell culture plate (Falcon, USA). The culture was performed in a carbon dioxide incubator at 37 ° C., 5% carbon dioxide, and 100% humidity. The liquid culture medium was serum-free Iskov modified Dulbecco medium (IMDM, manufactured by GIBCO-BRL, USA), 2% BSA, 10 μg / ml human insulin, 200 μg / ml transferrin, 40 μg / ml low density lipoprotein (US GIBCO-BRL). ^10-5 M2-mercaptoethanol, 50 ng / ml human SCF, 5 ng / ml human IL-3, 10 ng / ml human IL-6, 5 ng / ml human GM-CSF (manufactured by Intergen, USA), 3 U / ml What added Epo was used. Depending on conditions, 500 ng HSIg or 50 ng / ml MIP-1α (Intergen, USA) was added. 1 ml of this medium was added per well, and a half-volume medium was changed three times a week. After 2, 4, 6, and 8 weeks of culture, all cells were collected from the well into a 1.5 ml microtube using a cell scraper. The cells were sedimented by centrifugation, resuspended in 1 ml of fresh IMDM, the number of cells was counted using a hemocytometer, and a blood cell colony formation assay was performed at 5000 cells / ml.

  The hematopoietic colony formation assay uses Iskov methylcellulose complete ready mix (manufactured by Stem Cell Technologies, Canada), 1 ml each was planted on two 35 mm dishes (Falcon, USA) using a syringe, and cultured in a carbon dioxide incubator for 2 weeks. did. Blood cell colonies were counted as granulocyte monocyte colonies (CFU-GM) and erythroid colonies (BFU-E) under an inverted microscope, and the total number was taken as CFU-C. The number of CFU-C obtained here was multiplied by the number of cells determined with a hemocytometer, and the number of CFU-C per 1000 cells planted in liquid culture was determined. The results are shown in Table 1 for HDEXIg and in Table 2 for HSEXIg. The experiment was performed with n = 3, and the value was expressed as (mean value ± SD). Note that ND in the table indicates that no colonies could be detected.

  As a result, CFU-C was observed only up to 6 weeks in the absence of cytokines that maintained an undifferentiated state, and in the presence of MIP-1α, but also in the presence of HDEXIg or HSEXIg. It was. In comparison of MIP-1α with HDEXIg and HSEXIg, MIP-1α was observed to strongly suppress colony formation at 2 weeks of culture, but inhibition with HDEXIg and HSEXIg was not observed. In addition, it was observed that HDEXIg and HSEXIg were excellent in maintaining the CFU-C number at 6 weeks and 8 weeks of culture.

Example 12 Effects of HDEXIg and HSEXIg on liquid culture of blood undifferentiated cells LTC-IC In order to observe physiological effects of HDEXIg and HSEXIg on blood undifferentiated cells, cord blood CD34 positive cells were treated with HDEXIg or HSEXIg and existing cytokines. Then, liquid culture was performed in a serum-free medium for 2 weeks, and the increase or decrease in LTC-IC, which is considered to be the most undifferentiated blood cell group, was observed.

  Umbilical cord blood mononuclear cells CD34-positive cells isolated by the method described in Example 10 were cultured in 100,000 to 20,000 cells in the following medium for 2 weeks, and were divided into 4 groups: the pre-culture group, the HDEXIg presence group, the HSEXIg presence group, and the comparison group. The difference in the number of LTC-IC present in the experimental plot was examined.

The medium used for liquid culture was α-medium with 2% BSA, 10 μg / ml human insulin, 200 μg / ml transferrin, 40 μg / ml low density lipoprotein, 10 ⁻⁵ M2-mercaptoethanol, and further 100 ng / ml human SCF. A medium supplemented with 10 ng / ml human IL-3 and 100 ng / ml human IL-6 was used, HDEXIg or HSEXIg was added thereto at 1 μg / ml, and the above-mentioned human IgG1 was added to the comparison group at the same concentration.

  Preparation of human bone marrow stromal cell layer to be used for LTC-IC, and quantification of the frequency of LTC-IC by limiting dilution method is the method of Sutherland et al. (Blood, 74, 1563-1989, Proc. Natl. Acad. Sci. USA, 87, 3584, 1990).

That is, the LTC medium (MyeloCult, Stem Cell, Canada) supplemented with 1 μM hydrocortisone (manufactured by Upjohn, Japan) of the bone marrow mononuclear cells obtained in Example 10 that were not treated with the silica solution was added to 1 × 2 × 10 ⁷ cells. In 5 ml of Technologies (manufactured by Technologies) in a T-25 flask (manufactured by Falcon, USA) in a carbon dioxide incubator under an atmosphere of 37 ° C., 5% carbon dioxide and 100% humidity, formation of stromal cells as an adherent cell layer The culture was continued until it reached 80% or more of the bottom area, and then treated with a trypsin EDTA solution (manufactured by Cosmo Bio, Japan) and peeled off. About 2 × 10 ⁴ cells per well are placed in a 96-well plate (made by Becton Decktonson, USA), and the culture is continued again in the same medium to reconstitute stromal cells, and then 250 Kilovolt Peak X-rays are used. A product obtained by irradiating with 15 Gy and removing stromal cell proliferation and blood cells in the stromal cell was used as a stromal cell layer in the experiment.

When the cells of each experimental group cultured by the above method are used in the LTC-IC assay, 25 to 400 CD34 positive cell cells before culturing per well, and 625 to 20000 cells in each experimental group after culturing. In the 96-well plate formed with the above stromal cells, the cells were co-cultured in 16 wells for each dilution step. The culture medium was the medium used for stroma formation, and the conditions were 37 ° C., 5% carbon dioxide gas, carbon dioxide gas incubator under 100% humidity atmosphere for 5 weeks. Cells after culture were collected as floating cells and adherent cells per well, 0.9% methylcellulose, 30% fetal calf serum (FCS, ICN Biomedical Japan, Japan), 1% BSA, 10 ^{−5 in} α-medium. Transfer to semi-solid medium supplemented with M2-merkabutethanol, 100 ng / ml human SCF, 10 ng / ml human IL-3, 100 ng / ml human IL-6, 2 U / ml Epo, 10 ng / ml human G-CSF, 2 weeks After culturing, colony-forming cells were detected in the same manner as in Examples 10 and 11, and the number of wells in which colony-forming cells were present was detected. Based on this data, the frequency of LTC-IC was calculated according to the method of Taswell et al. (J. Immunol., 126, 1614-, 1981).

  The graph used for the calculation is shown in FIG. FIG. 4 is a graph of calculation after liquid culture. The vertical axis shows the ratio of wells where colonies did not appear in logarithm, and the horizontal axis shows the number of cells per well. The number of wells and cells in which colonies did not appear in each experimental plot were plotted, and a regression line was obtained by the method of least squares. The number of wells in which no colonies appeared was 0.37 (the reciprocal of the base of natural logarithm) The number of cells is calculated, and the reciprocal of the number of cells is the frequency of LTC-IC. Furthermore, the absolute number of LTC-IC was calculated from the initial cell number and the frequency of LTC-IC.

  As a result, 243 LTC-IC existed in 25,000 cells before liquid culture, and in the comparison plot, the number of cells increased to 1,5150,000 after 2 weeks of culture. Reduced to pieces. However, when cultured in a medium containing human delta-1 or HDEXIg or human selete-1 or HSEXIg, the number of cells increased to almost the same number as 1,310,000 and 1,140,000, respectively. However, the number of LTC-ICs was reduced to 115 and 53, respectively. From this result, it was clarified that the polypeptide of the present invention, particularly human delta-1, has the effect of maintaining LTC-IC in liquid culture.

Example 13 Binding of HDEXIg and HSEXIg to blood cell lines Using the property that Notch ligand molecules specifically bind to Notch receptors, the binding of Notch ligand molecules to various blood cell lines was examined.

  The cell lines are blood cell lines such as Jurkat (ATCC TIB-152), Namalwa (ATCC CRL-1432), HL-60 (ATCC CRL-1964), K562 (ATCC CCL-243), THP-1 (ATCCTIB-202), UT-7 (Komatsu et al., Cancer Res., 51, 341-348, 1991), Mo7e (Avanzi et al., Br. J. Haematol., 69, 359-, 1988), CMK (Sato et al. , Exp. Hematol., 15, 495-502, 1987). The culture medium of these cell lines was the medium described in each of the cited references or ATCC CELL LINES & HYBRIDOMAS 8th ed (1994).

1 × 10 ⁶ cells were suspended in Hank's solution containing 2% FCS and 10 mM Hepes, HDEXIg or HSEXIg was added at 1 μg / ml, and left overnight at 4 ° C. After washing twice with the same Hanks solution, 1 μg / ml of PE-labeled goat anti-human IgG monoclonal antibody was added, left on ice for 30 minutes, washed twice with Hanks solution, and finally suspended in 1 ml Hanks solution. For analysis. The measurement was performed with a flow cytometer (FACSCalibur). Moreover, what was dye | stained with this human IgG1 instead of HDEXIg and HSEXIg was used as control.

  The results are shown in FIG. The vertical axis represents the number of cells, and the horizontal axis represents the fluorescence intensity. Those stained with HDEXIg and HSEXIg are indicated by solid lines, those stained with control human IgG1 are indicated by broken lines, the left column indicates HDEXIg, and the right column indicates HSEXIg. From these results, Jurkat reacted, Namalwa unreacted, HL-60 unreacted, K562 unreacted, THP-1 unreacted, UT-7 reacted, Mo7e unreacted, and CMK reacted. Moreover, since these results were the same for HDEXIg and HSEXIg, it was clear that the same molecules were recognized, and that these cells could be sorted.

FIG. 1 is an alignment of DSL domains of Notch ligand molecules identified in various organisms containing the molecule of the present invention. FIG. 2 shows suppression of colony formation of blood undifferentiated cells by the molecule of the present invention. FIG. 3 shows the concentration dependence of the colony formation inhibitory action of blood undifferentiated cells of the molecule of the present invention. FIG. 4 shows a graph when calculating LTC-IC after liquid culture using the molecule of the present invention. FIG. 5 shows cells stained with the molecule of the present invention.

[Sequence Listing]
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Arg Asx Asp Xaa Phe Gly His Xaa Xaa Cys Xaa Xaa Xaa Gly Xaa Xaa
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Ser Ala Phe Ser Asn Pro Ile Arg Phe Pro Phe Gly Phe Thr Trp Pro
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His His Lys Pro Cys Lys Asn Gly Ala Thr Cys Thr Asn Thr Gly Gln
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Phe Tyr Gly Lys Ile Cys Glu Leu Ser Ala Met Thr Cys Ala Asp Gly
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Ile Asp Tyr Cys Ser Ser Pro Pro Cys Ser Asn Gly Ala Lys Cys Val
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Asp Leu Gly Asp Ala Tyr Leu Cys Arg Cys Gln Ala Gly Phe Ser Gly
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Arg His Cys Asp Asp Asn Val Asp Asp Cys Ala Ser Ser Pro Cys Ala
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Arg Tyr Val Cys Glu Cys Ala Arg Gly Tyr Gly Gly Pro Asn Cys Gln
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Phe Leu Leu Pro Glu Leu Pro Pro Gly Pro Ala Val Val Asp Leu Thr
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Glu Lys Leu Glu Gly Gln Gly Gly
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Ser Val Ser Pro Glu Pro Pro Cys Thr Tyr Gly Ser Ala Val Thr Pro
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Val Leu Gly Val Asp Ser Phe Ser Leu Pro Asp Gly Gly Gly Ala Asp
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Ser Ala Phe Ser Asn Pro Ile Arg Phe Pro Phe Gly Phe Thr Trp Pro
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Gly Thr Phe Ser Leu Ile Ile Glu Ala Leu His Thr Asp Ser Pro Asp
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Asp Leu Ala Thr Glu Asn Pro Glu Arg Leu Ile Ser Arg Leu Ala Thr
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Gln Arg His Leu Thr Val Gly Glu Glu Trp Ser Gln Asp Leu His Ser
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Ser Gly Arg Thr Asp Leu Lys Tyr Ser Tyr Arg Phe Val Cys Asp Glu
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His Tyr Tyr Gly Glu Gly Cys Ser Val Phe Cys Arg Pro Arg Asp Asp
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His His Lys Pro Cys Lys Asn Gly Ala Thr Cys Thr Asn Thr Gly Gln
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Glu Leu Gly Ile Asp Glu Cys Asp Pro Ser Pro Cys Lys Asn Gly Gly
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Ile Asp Tyr Cys Ser Ser Pro Pro Cys Ser Asn Gly Ala Lys Cys Val
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Asp Leu Gly Asp Ala Tyr Leu Cys Arg Cys Gln Ala Gly Phe Ser Gly
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Arg His Cys Asp Asp Asn Val Asp Asp Cys Ala Ser Ser Pro Cys Ala
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Asn Gly Gly Thr Cys Arg Asp Gly Val Asn Asp Phe Ser Cys Thr Cys
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Pro Pro Gly Tyr Thr Gly Arg Asn Cys Ser Ala Pro Val Ser Arg Cys
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Arg Tyr Val Cys Glu Cys Ala Arg Gly Tyr Gly Gly Pro Asn Cys Gln
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Phe Leu Leu Pro Glu Leu Pro Pro Gly Pro Ala Val Val Asp Leu Thr
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Glu Lys Leu Glu Gly Gln Gly Gly Pro Phe Pro Trp Val Ala Val Cys
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Ala Gly Val Ile Leu Val Leu Met Leu Leu Leu Gly Cys Ala Ala Val
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Val Val Cys Val Arg Leu Arg Leu Gln Lys His Arg Pro Pro Ala Asp
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Pro Cys Arg Gly Glu Thr Glu Thr Met Asn Asn Leu Ala Asn Cys Gln
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Arg Glu Lys Asp Ile Ser Val Ser Ile Ile Gly Ala Thr Gln Ile Lys
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Asn Thr Asn Lys Lys Ala Asp Phe His Gly Asp His Ser Ala Asp Lys
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Asn Gly Phe Lys Ala Arg Tyr Pro Ala Val Asp Tyr Asn Leu Val Gln
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Asp Thr Lys Cys Gln Pro Gln Gly Ser Ser Gly Glu Glu Lys Gly Thr
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Pro Thr Thr Leu Arg Gly Gly Glu Ala Ser Glu Arg Lys Arg Pro Asp
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Ser Gly Cys Ser Thr Ser Lys Asp Thr Lys Tyr Gln Ser Val Tyr Val
675 680 685
Ile Ser Glu Glu Lys Asp Glu Cys Val Ile Ala Thr Glu Val
690 695 700
<210> 5
<211> 198
<212> PRT
<213> Homo sapiens
<400> 5
Ser Gly Gln Phe Glu Leu Glu Ile Leu Ser Met Gln Asn Val Asn Gly
1 5 10 15
Glu Leu Gln Asn Gly Asn Cys Cys Gly Gly Ala Arg Asn Pro Gly Asp
20 25 30
Arg Lys Cys Thr Arg Asp Glu Cys Asp Thr Tyr Phe Lys Val Cys Leu
35 40 45
Lys Glu Tyr Gln Ser Arg Val Thr Ala Gly Gly Pro Cys Ser Phe Gly
50 55 60
Ser Gly Ser Thr Pro Val Ile Gly Gly Asn Thr Phe Asn Leu Lys Ala
65 70 75 80
Ser Arg Gly Asn Asp Arg Asn Arg Ile Val Leu Pro Phe Ser Phe Ala
85 90 95
Trp Pro Arg Ser Tyr Thr Leu Leu Val Glu Ala Trp Asp Ser Ser Asn
100 105 110
Asp Thr Val Gln Pro Asp Ser Ile Ile Glu Lys Ala Ser His Ser Gly
115 120 125
Met Ile Asn Pro Ser Arg Gln Trp Gln Thr Leu Lys Gln Asn Thr Gly
130 135 140
Val Ala His Phe Glu Tyr Gln Ile Arg Val Thr Cys Asp Asp Tyr Tyr
145 150 155 160
Tyr Gly Phe Gly Cys Asn Lys Phe Cys Arg Pro Arg Asp Asp Phe Phe
165 170 175
Gly His Tyr Ala Cys Asp Gln Asn Gly Asn Lys Thr Cys Met Glu Gly
180 185 190
Trp Met Gly Pro Glu Cys
195
<210> 6
<211> 1036
<212> PRT
<213> Homo sapiens
<400> 6
Ser Gly Gln Phe Glu Leu Glu Ile Leu Ser Met Gln Asn Val Asn Gly
1 5 10 15
Glu Leu Gln Asn Gly Asn Cys Cys Gly Gly Ala Arg Asn Pro Gly Asp
20 25 30
Arg Lys Cys Thr Arg Asp Glu Cys Asp Thr Tyr Phe Lys Val Cys Leu
35 40 45
Lys Glu Tyr Gln Ser Arg Val Thr Ala Gly Gly Pro Cys Ser Phe Gly
50 55 60
Ser Gly Ser Thr Pro Val Ile Gly Gly Asn Thr Phe Asn Leu Lys Ala
65 70 75 80
Ser Arg Gly Asn Asp Arg Asn Arg Ile Val Leu Pro Phe Ser Phe Ala
85 90 95
Trp Pro Arg Ser Tyr Thr Leu Leu Val Glu Ala Trp Asp Ser Ser Asn
100 105 110
Asp Thr Val Gln Pro Asp Ser Ile Ile Glu Lys Ala Ser His Ser Gly
115 120 125
Met Ile Asn Pro Ser Arg Gln Trp Gln Thr Leu Lys Gln Asn Thr Gly
130 135 140
Val Ala His Phe Glu Tyr Gln Ile Arg Val Thr Cys Asp Asp Tyr Tyr
145 150 155 160
Tyr Gly Phe Gly Cys Asn Lys Phe Cys Arg Pro Arg Asp Asp Phe Phe
165 170 175
Gly His Tyr Ala Cys Asp Gln Asn Gly Asn Lys Thr Cys Met Glu Gly
180 185 190
Trp Met Gly Pro Glu Cys Asn Arg Ala Ile Cys Arg Gln Gly Cys Ser
195 200 205
Pro Lys His Gly Ser Cys Lys Leu Pro Gly Asp Cys Arg Cys Gln Tyr
210 215 220
Gly Trp Gln Gly Leu Tyr Cys Asp Lys Cys Ile Pro His Pro Gly Cys
225 230 235 240
Val His Gly Ile Cys Asn Glu Pro Trp Gln Cys Leu Cys Glu Thr Asn
245 250 255
Trp Gly Gly Gln Leu Cys Asp Lys Asp Leu Asn Tyr Cys Gly Thr His
260 265 270
Gln Pro Cys Leu Asn Gly Gly Thr Cys Ser Asn Thr Gly Pro Asp Lys
275 280 285
Tyr Gln Cys Ser Cys Pro Glu Gly Tyr Ser Gly Pro Asn Cys Glu Ile
290 295 300
Ala Glu His Ala Cys Leu Ser Asp Pro Cys His Asn Arg Gly Ser Cys
305 310 315 320
Lys Glu Thr Ser Leu Gly Phe Glu Cys Glu Cys Ser Pro Gly Trp Thr
325 330 335
Gly Pro Thr Cys Ser Thr Asn Ile Asp Asp Cys Ser Pro Asn Asn Cys
340 345 350
Ser His Gly Gly Thr Cys Gln Asp Leu Val Asn Gly Phe Lys Cys Val
355 360 365
Cys Pro Pro Gln Trp Thr Gly Lys Thr Cys Gln Leu Asp Ala Asn Glu
370 375 380
Cys Glu Ala Lys Pro Cys Val Asn Ala Lys Ser Cys Lys Asn Leu Ile
385 390 395 400
Ala Ser Tyr Tyr Cys Asp Cys Leu Pro Gly Trp Met Gly Gln Asn Cys
405 410 415
Asp Ile Asn Ile Asn Asp Cys Leu Gly Gln Cys Gln Asn Asp Ala Ser
420 425 430
Cys Arg Asp Leu Val Asn Gly Tyr Arg Cys Ile Cys Pro Pro Gly Tyr
435 440 445
Ala Gly Asp His Cys Glu Arg Asp Ile Asp Glu Cys Ala Ser Asn Pro
450 455 460
Cys Leu Asn Gly Gly His Cys Gln Asn Glu Ile Asn Arg Phe Gln Cys
465 470 475 480
Leu Cys Pro Thr Gly Phe Ser Gly Asn Leu Cys Gln Leu Asp Ile Asp
485 490 495
Tyr Cys Glu Pro Asn Pro Cys Gln Asn Gly Ala Gln Cys Tyr Asn Arg
500 505 510
Ala Ser Asp Tyr Phe Cys Lys Cys Pro Glu Asp Tyr Glu Gly Lys Asn
515 520 525
Cys Ser His Leu Lys Asp His Cys Arg Thr Thr Pro Cys Glu Val Ile
530 535 540
Asp Ser Cys Thr Val Ala Met Ala Ser Asn Asp Thr Pro Glu Gly Val
545 550 555 560
Arg Tyr Ile Ser Ser Asn Val Cys Gly Pro His Gly Lys Cys Lys Ser
565 570 575
Gln Ser Gly Gly Lys Phe Thr Cys Asp Cys Asn Lys Gly Phe Thr Gly
580 585 590
Thr Tyr Cys His Glu Asn Ile Asn Asp Cys Glu Ser Asn Pro Cys Arg
595 600 605
Asn Gly Gly Thr Cys Ile Asp Gly Val Asn Ser Tyr Lys Cys Ile Cys
610 615 620
Ser Asp Gly Trp Glu Gly Ala Tyr Cys Glu Thr Asn Ile Asn Asp Cys
625 630 635 640
Ser Gln Asn Pro Cys His Asn Gly Gly Thr Cys Arg Asp Leu Val Asn
645 650 655
Asp Phe Tyr Cys Asp Cys Lys Asn Gly Trp Lys Gly Lys Thr Cys His
660 665 670
Ser Arg Asp Ser Gln Cys Asp Glu Ala Thr Cys Asn Asn Gly Gly Thr
675 680 685
Cys Tyr Asp Glu Gly Asp Ala Phe Lys Cys Met Cys Pro Gly Gly Trp
690 695 700
Glu Gly Thr Thr Cys Asn Ile Ala Arg Asn Ser Ser Cys Leu Pro Asn
705 710 715 720
Pro Cys His Asn Gly Gly Thr Cys Val Val Asn Gly Glu Ser Phe Thr
725 730 735
Cys Val Cys Lys Glu Gly Trp Glu Gly Pro Ile Cys Ala Gln Asn Thr
740 745 750
Asn Asp Cys Ser Pro His Pro Cys Tyr Asn Ser Gly Thr Cys Val Asp
755 760 765
Gly Asp Asn Trp Tyr Arg Cys Glu Cys Ala Pro Gly Phe Ala Gly Pro
770 775 780
Asp Cys Arg Ile Asn Ile Asn Glu Cys Gln Ser Ser Pro Cys Ala Phe
785 790 795 800
Gly Ala Thr Cys Val Asp Glu Ile Asn Gly Tyr Arg Cys Val Cys Pro
805 810 815
Pro Gly His Ser Gly Ala Lys Cys Gln Glu Val Ser Gly Arg Pro Cys
820 825 830
Ile Thr Met Gly Ser Val Ile Pro Asp Gly Ala Lys Trp Asp Asp Asp
835 840 845
Cys Asn Thr Cys Gln Cys Leu Asn Gly Arg Ile Ala Cys Ser Lys Val
850 855 860
Trp Cys Gly Pro Arg Pro Cys Leu Leu His Lys Gly His Ser Glu Cys
865 870 875 880
Pro Ser Gly Gln Ser Cys Ile Pro Ile Leu Asp Asp Gln Cys Phe Val
885 890 895
His Pro Cys Thr Gly Val Gly Glu Cys Arg Ser Ser Ser Leu Gln Pro
900 905 910
Val Lys Thr Lys Cys Thr Ser Asp Ser Tyr Tyr Gln Asp Asn Cys Ala
915 920 925
Asn Ile Thr Phe Thr Phe Asn Lys Glu Met Met Ser Pro Gly Leu Thr
930 935 940
Thr Glu His Ile Cys Ser Glu Leu Arg Asn Leu Asn Ile Leu Lys Asn
945 950 955 960
Val Ser Ala Glu Tyr Ser Ile Tyr Ile Ala Cys Glu Pro Ser Pro Ser
965 970 975
Ala Asn Asn Glu Ile His Val Ala Ile Ser Ala Glu Asp Ile Arg Asp
980 985 990
Asp Gly Asn Pro Ile Lys Glu Ile Thr Asp Lys Ile Ile Asp Leu Val
995 1000 1005
Ser Lys Arg Asp Gly Asn Ser Ser Leu Ile Ala Ala Val Ala Glu Val
1010 1015 1020
Arg Val Gln Arg Arg Pro Leu Lys Asn Arg Thr Asp
1025 1030 1035
<210> 7
<211> 1187
<212> PRT
<213> Homo sapiens
<400> 7
Ser Gly Gln Phe Glu Leu Glu Ile Leu Ser Met Gln Asn Val Asn Gly
1 5 10 15
Glu Leu Gln Asn Gly Asn Cys Cys Gly Gly Ala Arg Asn Pro Gly Asp
20 25 30
Arg Lys Cys Thr Arg Asp Glu Cys Asp Thr Tyr Phe Lys Val Cys Leu
35 40 45
Lys Glu Tyr Gln Ser Arg Val Thr Ala Gly Gly Pro Cys Ser Phe Gly
50 55 60
Ser Gly Ser Thr Pro Val Ile Gly Gly Asn Thr Phe Asn Leu Lys Ala
65 70 75 80
Ser Arg Gly Asn Asp Arg Asn Arg Ile Val Leu Pro Phe Ser Phe Ala
85 90 95
Trp Pro Arg Ser Tyr Thr Leu Leu Val Glu Ala Trp Asp Ser Ser Asn
100 105 110
Asp Thr Val Gln Pro Asp Ser Ile Ile Glu Lys Ala Ser His Ser Gly
115 120 125
Met Ile Asn Pro Ser Arg Gln Trp Gln Thr Leu Lys Gln Asn Thr Gly
130 135 140
Val Ala His Phe Glu Tyr Gln Ile Arg Val Thr Cys Asp Asp Tyr Tyr
145 150 155 160
Tyr Gly Phe Gly Cys Asn Lys Phe Cys Arg Pro Arg Asp Asp Phe Phe
165 170 175
Gly His Tyr Ala Cys Asp Gln Asn Gly Asn Lys Thr Cys Met Glu Gly
180 185 190
Trp Met Gly Pro Glu Cys Asn Arg Ala Ile Cys Arg Gln Gly Cys Ser
195 200 205
Pro Lys His Gly Ser Cys Lys Leu Pro Gly Asp Cys Arg Cys Gln Tyr
210 215 220
Gly Trp Gln Gly Leu Tyr Cys Asp Lys Cys Ile Pro His Pro Gly Cys
225 230 235 240
Val His Gly Ile Cys Asn Glu Pro Trp Gln Cys Leu Cys Glu Thr Asn
245 250 255
Trp Gly Gly Gln Leu Cys Asp Lys Asp Leu Asn Tyr Cys Gly Thr His
260 265 270
Gln Pro Cys Leu Asn Gly Gly Thr Cys Ser Asn Thr Gly Pro Asp Lys
275 280 285
Tyr Gln Cys Ser Cys Pro Glu Gly Tyr Ser Gly Pro Asn Cys Glu Ile
290 295 300
Ala Glu His Ala Cys Leu Ser Asp Pro Cys His Asn Arg Gly Ser Cys
305 310 315 320
Lys Glu Thr Ser Leu Gly Phe Glu Cys Glu Cys Ser Pro Gly Trp Thr
325 330 335
Gly Pro Thr Cys Ser Thr Asn Ile Asp Asp Cys Ser Pro Asn Asn Cys
340 345 350
Ser His Gly Gly Thr Cys Gln Asp Leu Val Asn Gly Phe Lys Cys Val
355 360 365
Cys Pro Pro Gln Trp Thr Gly Lys Thr Cys Gln Leu Asp Ala Asn Glu
370 375 380
Cys Glu Ala Lys Pro Cys Val Asn Ala Lys Ser Cys Lys Asn Leu Ile
385 390 395 400
Ala Ser Tyr Tyr Cys Asp Cys Leu Pro Gly Trp Met Gly Gln Asn Cys
405 410 415
Asp Ile Asn Ile Asn Asp Cys Leu Gly Gln Cys Gln Asn Asp Ala Ser
420 425 430
Cys Arg Asp Leu Val Asn Gly Tyr Arg Cys Ile Cys Pro Pro Gly Tyr
435 440 445
Ala Gly Asp His Cys Glu Arg Asp Ile Asp Glu Cys Ala Ser Asn Pro
450 455 460
Cys Leu Asn Gly Gly His Cys Gln Asn Glu Ile Asn Arg Phe Gln Cys
465 470 475 480
Leu Cys Pro Thr Gly Phe Ser Gly Asn Leu Cys Gln Leu Asp Ile Asp
485 490 495
Tyr Cys Glu Pro Asn Pro Cys Gln Asn Gly Ala Gln Cys Tyr Asn Arg
500 505 510
Ala Ser Asp Tyr Phe Cys Lys Cys Pro Glu Asp Tyr Glu Gly Lys Asn
515 520 525
Cys Ser His Leu Lys Asp His Cys Arg Thr Thr Pro Cys Glu Val Ile
530 535 540
Asp Ser Cys Thr Val Ala Met Ala Ser Asn Asp Thr Pro Glu Gly Val
545 550 555 560
Arg Tyr Ile Ser Ser Asn Val Cys Gly Pro His Gly Lys Cys Lys Ser
565 570 575
Gln Ser Gly Gly Lys Phe Thr Cys Asp Cys Asn Lys Gly Phe Thr Gly
580 585 590
Thr Tyr Cys His Glu Asn Ile Asn Asp Cys Glu Ser Asn Pro Cys Arg
595 600 605
Asn Gly Gly Thr Cys Ile Asp Gly Val Asn Ser Tyr Lys Cys Ile Cys
610 615 620
Ser Asp Gly Trp Glu Gly Ala Tyr Cys Glu Thr Asn Ile Asn Asp Cys
625 630 635 640
Ser Gln Asn Pro Cys His Asn Gly Gly Thr Cys Arg Asp Leu Val Asn
645 650 655
Asp Phe Tyr Cys Asp Cys Lys Asn Gly Trp Lys Gly Lys Thr Cys His
660 665 670
Ser Arg Asp Ser Gln Cys Asp Glu Ala Thr Cys Asn Asn Gly Gly Thr
675 680 685
Cys Tyr Asp Glu Gly Asp Ala Phe Lys Cys Met Cys Pro Gly Gly Trp
690 695 700
Glu Gly Thr Thr Cys Asn Ile Ala Arg Asn Ser Ser Cys Leu Pro Asn
705 710 715 720
Pro Cys His Asn Gly Gly Thr Cys Val Val Asn Gly Glu Ser Phe Thr
725 730 735
Cys Val Cys Lys Glu Gly Trp Glu Gly Pro Ile Cys Ala Gln Asn Thr
740 745 750
Asn Asp Cys Ser Pro His Pro Cys Tyr Asn Ser Gly Thr Cys Val Asp
755 760 765
Gly Asp Asn Trp Tyr Arg Cys Glu Cys Ala Pro Gly Phe Ala Gly Pro
770 775 780
Asp Cys Arg Ile Asn Ile Asn Glu Cys Gln Ser Ser Pro Cys Ala Phe
785 790 795 800
Gly Ala Thr Cys Val Asp Glu Ile Asn Gly Tyr Arg Cys Val Cys Pro
805 810 815
Pro Gly His Ser Gly Ala Lys Cys Gln Glu Val Ser Gly Arg Pro Cys
820 825 830
Ile Thr Met Gly Ser Val Ile Pro Asp Gly Ala Lys Trp Asp Asp Asp
835 840 845
Cys Asn Thr Cys Gln Cys Leu Asn Gly Arg Ile Ala Cys Ser Lys Val
850 855 860
Trp Cys Gly Pro Arg Pro Cys Leu Leu His Lys Gly His Ser Glu Cys
865 870 875 880
Pro Ser Gly Gln Ser Cys Ile Pro Ile Leu Asp Asp Gln Cys Phe Val
885 890 895
His Pro Cys Thr Gly Val Gly Glu Cys Arg Ser Ser Ser Leu Gln Pro
900 905 910
Val Lys Thr Lys Cys Thr Ser Asp Ser Tyr Tyr Gln Asp Asn Cys Ala
915 920 925
Asn Ile Thr Phe Thr Phe Asn Lys Glu Met Met Ser Pro Gly Leu Thr
930 935 940
Thr Glu His Ile Cys Ser Glu Leu Arg Asn Leu Asn Ile Leu Lys Asn
945 950 955 960
Val Ser Ala Glu Tyr Ser Ile Tyr Ile Ala Cys Glu Pro Ser Pro Ser
965 970 975
Ala Asn Asn Glu Ile His Val Ala Ile Ser Ala Glu Asp Ile Arg Asp
980 985 990
Asp Gly Asn Pro Ile Lys Glu Ile Thr Asp Lys Ile Ile Asp Leu Val
995 1000 1005
Ser Lys Arg Asp Gly Asn Ser Ser Leu Ile Ala Ala Val Ala Glu Val
1010 1015 1020
Arg Val Gln Arg Arg Pro Leu Lys Asn Arg Thr Asp Phe Leu Val Pro
1025 1030 1035 1040
Leu Leu Ser Ser Val Leu Thr Val Ala Trp Ile Cys Cys Leu Val Thr
1045 1050 1055
Ala Phe Tyr Trp Cys Leu Arg Lys Arg Arg Lys Pro Gly Ser His Thr
1060 1065 1070
His Ser Ala Ser Glu Asp Asn Thr Thr Asn Asn Val Arg Glu Gln Leu
1075 1080 1085
Asn Gln Ile Lys Asn Pro Ile Glu Lys His Gly Ala Asn Thr Val Pro
1090 1095 1100
Ile Lys Asp Tyr Glu Asn Lys Asn Ser Lys Met Ser Lys Ile Arg Thr
1105 1110 1115 1120
His Asn Ser Glu Val Glu Glu Asp Asp Met Asp Lys His Gln Gln Lys
1125 1130 1135
Ala Arg Phe Ala Lys Gln Pro Ala Tyr Thr Leu Val Asp Arg Glu Glu
1140 1145 1150
Lys Pro Pro Asn Gly Thr Pro Thr Lys His Pro Asn Trp Thr Asn Lys
1155 1160 1165
Gln Asp Asn Arg Asp Leu Glu Ser Ala Gln Ser Leu Asn Arg Met Glu
1170 1175 1180
Tyr Ile Val
1185
<210> 8
<211> 2663
<212> DNA
<213> Homo sapiens
<400> 8
cttgggaaga ggcggagacc ggcttttaaa gaaagaagtc ctgggtcctg cggtctgggg 60
cgaggcaagg gcgcttttct gcccacgctc cccgtggccc atcgatcccc cgcgcgtccg 120
ccgctgttct aaggagagaa gtgggggccc cccaggctcg cgcgtggagc gaagcagc 178
atg ggc agt cgg tgc gcg ctg gcc ctg gcg gtg ctc tcg gcc ttg ctg 226
Met Gly Ser Arg Cys Ala Leu Ala Leu Ala Val Leu Ser Ala Leu Leu
1 5 10 15
tgt cag gtc tgg agc tct ggg gtg ttc gaa ctg aag ctg cag gag ttc 274
Cys Gln Val Trp Ser Ser Gly Val Phe Glu Leu Lys Leu Gln Glu Phe
20 25 30
gtc aac aag aag ggg ctg ctg ggg aac cgc aac tgc tgc cgc ggg ggc 322
Val Asn Lys Lys Gly Leu Leu Gly Asn Arg Asn Cys Cys Arg Gly Gly
35 40 45
gcg ggg cca ccg ccg tgc gcc tgc cgg acc ttc ttc cgc gtg tgc ctc 370
Ala Gly Pro Pro Pro Cys Ala Cys Arg Thr Phe Phe Arg Val Cys Leu
50 55 60
aag cac tac cag gcc agc gtg tcc ccc gag ccg ccc tgc acc tac ggc 418
Lys His Tyr Gln Ala Ser Val Ser Pro Glu Pro Pro Cys Thr Tyr Gly
65 70 75 80
agc gcc gtc acc ccc gtg ctg ggc gtc gac tcc ttc agt ctg ccc gac 466
Ser Ala Val Thr Pro Val Leu Gly Val Asp Ser Phe Ser Leu Pro Asp
85 90 95
ggc ggg ggc gcc gac tcc gcg ttc agc aac ccc atc cgc ttc ccc ttc 514
Gly Gly Gly Ala Asp Ser Ala Phe Ser Asn Pro Ile Arg Phe Pro Phe
100 105 110
ggc ttc acc tgg ccg ggc acc ttc tct ctg att att gaa gct ctc cac 562
Gly Phe Thr Trp Pro Gly Thr Phe Ser Leu Ile Ile Glu Ala Leu His
115 120 125
aca gat tct cct gat gac ctc gca aca gaa aac cca gaa aga ctc atc 610
Thr Asp Ser Pro Asp Asp Leu Ala Thr Glu Asn Pro Glu Arg Leu Ile
130 135 140
agc cgc ctg gcc acc cag agg cac ctg acg gtg ggc gag gag tgg tcc 658
Ser Arg Leu Ala Thr Gln Arg His Leu Thr Val Gly Glu Glu Trp Ser
145 150 155 160
cag gac ctg cac agc agc ggc cgc acg gac ctc aag tac tcc tac cgc 706
Gln Asp Leu His Ser Ser Gly Arg Thr Asp Leu Lys Tyr Ser Tyr Arg
165 170 175
ttc gtg tgt gac gaa cac tac tac gga gag ggc tgc tcc gtt ttc tgc 754
Phe Val Cys Asp Glu His Tyr Tyr Gly Glu Gly Cys Ser Val Phe Cys
180 185 190
cgt ccc cgg gac gat gcc ttc ggc cac ttc acc tgt ggg gag cgt ggg 802
Arg Pro Arg Asp Asp Ala Phe Gly His Phe Thr Cys Gly Glu Arg Gly
195 200 205
gag aaa gtg tgc aac cct ggc tgg aaa ggg ccc tac tgc aca gag ccg 850
Glu Lys Val Cys Asn Pro Gly Trp Lys Gly Pro Tyr Cys Thr Glu Pro
210 215 220
atc tgc ctg cct gga tgt gat gag cag cat gga ttt tgt gac aaa cca 898
Ile Cys Leu Pro Gly Cys Asp Glu Gln His Gly Phe Cys Asp Lys Pro
225 230 235 240
ggg gaa tgc aag tgc aga gtg ggc tgg cag ggc cgg tac tgt gac gag 946
Gly Glu Cys Lys Cys Arg Val Gly Trp Gln Gly Arg Tyr Cys Asp Glu
245 250 255
tgt atc cgc tat cca ggc tgt ctc cat ggc acc tgc cag cag ccc tgg 994
Cys Ile Arg Tyr Pro Gly Cys Leu His Gly Thr Cys Gln Gln Pro Trp
260 265 270
cag tgc aac tgc cag gaa ggc tgg ggg ggc ctt ttc tgc aac cag gac 1042
Gln Cys Asn Cys Gln Glu Gly Trp Gly Gly Leu Phe Cys Asn Gln Asp
275 280 285
ctg aac tac tgc aca cac cat aag ccc tgc aag aat gga gcc acc tgc 1090
Leu Asn Tyr Cys Thr His His Lys Pro Cys Lys Asn Gly Ala Thr Cys
290 295 300
acc aac acg ggc cag ggg agc tac act tgc tct tgc cgg cct ggg tac 1138
Thr Asn Thr Gly Gln Gly Ser Tyr Thr Cys Ser Cys Arg Pro Gly Tyr
305 310 315 320
aca ggt gcc acc tgc gag ctg ggg att gac gag tgt gac ccc agc cct 1186
Thr Gly Ala Thr Cys Glu Leu Gly Ile Asp Glu Cys Asp Pro Ser Pro
325 330 335
tgt aag aac gga ggg agc tgc acg gat ctc gag aac agc tac tcc tgt 1234
Cys Lys Asn Gly Gly Ser Cys Thr Asp Leu Glu Asn Ser Tyr Ser Cys
340 345 350
acc tgc cca ccc ggc ttc tac ggc aaa atc tgt gaa ttg agt gcc atg 1282
Thr Cys Pro Pro Gly Phe Tyr Gly Lys Ile Cys Glu Leu Ser Ala Met
355 360 365
acc tgt gcg gac ggc cct tgc ttt aac ggg ggt cgg tgc tca gac agc 1330
Thr Cys Ala Asp Gly Pro Cys Phe Asn Gly Gly Arg Cys Ser Asp Ser
370 375 380
ccc gat gga ggg tac agc tgc cgc tgc ccc gtg ggc tac tcc ggc ttc 1378
Pro Asp Gly Gly Tyr Ser Cys Arg Cys Pro Val Gly Tyr Ser Gly Phe
385 390 395 400
aac tgt gag aag aaa att gac tac tgc agc tct tca ccc tgt tct aat 1426
Asn Cys Glu Lys Lys Ile Asp Tyr Cys Ser Ser Ser Pro Cys Ser Asn
405 410 415
ggt gcc aag tgt gtg gac ctc ggt gat gcc tac ctg tgc cgc tgc cag 1474
Gly Ala Lys Cys Val Asp Leu Gly Asp Ala Tyr Leu Cys Arg Cys Gln
420 425 430
gcc ggc ttc tcg ggg agg cac tgt gac gac aac gtg gac gac tgc gcc 1522
Ala Gly Phe Ser Gly Arg His Cys Asp Asp Asn Val Asp Asp Cys Ala
435 440 445
tcc tcc ccg tgc gcc aac ggg ggc acc tgc cgg gat ggc gtg aac gac 1570
Ser Ser Pro Cys Ala Asn Gly Gly Thr Cys Arg Asp Gly Val Asn Asp
450 455 460
ttc tcc tgc acc tgc ccg cct ggc tac acg ggc agg aac tgc agt gcc 1618
Phe Ser Cys Thr Cys Pro Pro Gly Tyr Thr Gly Arg Asn Cys Ser Ala
465 470 475 480
ccc gtc agc agg tgc gag cac gca ccc tgc cac aat ggg gcc acc tgc 1666
Pro Val Ser Arg Cys Glu His Ala Pro Cys His Asn Gly Ala Thr Cys
485 490 495
cac gag agg ggc cac cgc tat gtg tgc gag tgt gcc cga ggc tac ggg 1714
His Glu Arg Gly His Arg Tyr Val Cys Glu Cys Ala Arg Gly Tyr Gly
500 505 510
ggt ccc aac tgc cag ttc ctg ctc ccc gag ctg ccc ccg ggc cca gcg 1762
Gly Pro Asn Cys Gln Phe Leu Leu Pro Glu Leu Pro Pro Gly Pro Ala
515 520 525
gtg gtg gac ctc act gag aag cta gag ggc cag ggc ggg cca ttc ccc 1810
Val Val Asp Leu Thr Glu Lys Leu Glu Gly Gln Gly Gly Pro Phe Pro
530 535 540
tgg gtg gcc gtg tgc gcc ggg gtc atc ctt gtc ctc atg ctg ctg ctg 1858
Trp Val Ala Val Cys Ala Gly Val Ile Leu Val Leu Met Leu Leu Leu
545 550 555 560
ggc tgt gcc gct gtg gtg gtc tgc gtc cgg ctg agg ctg cag aag cac 1906
Gly Cys Ala Ala Val Val Val Cys Val Arg Leu Arg Leu Gln Lys His
565 570 575
cgg ccc cca gcc gac ccc tgc cgg ggg gag acg gag acc atg aac aac 1954
Arg Pro Pro Ala Asp Pro Cys Arg Gly Glu Thr Glu Thr Met Asn Asn
580 585 590
ctg gcc aac tgc cag cgt gag aag gac atc tca gtc agc atc atc ggg 2002
Leu Ala Asn Cys Gln Arg Glu Lys Asp Ile Ser Val Ser Ile Ile Gly
595 600 605
gcc acg cag atc aag aac acc aac aag aag gcg gac ttc cac ggg gac 2050
Ala Thr Gln Ile Lys Asn Thr Asn Lys Lys Ala Asp Phe His Gly Asp
610 615 620
cac agc gcc gac aag aat ggc ttc aag gcc cgc tac cca gcg gtg gac 2098
His Ser Ala Asp Lys Asn Gly Phe Lys Ala Arg Tyr Pro Ala Val Asp
625 630 635 640
tat aac ctc gtg cag gac ctc aag ggt gac gac acc gcc gtc agg gac 2146
Tyr Asn Leu Val Gln Asp Leu Lys Gly Asp Asp Thr Ala Val Arg Asp
645 650 655
gcg cac agc aag cgt gac acc aag tgc cag ccc cag ggc tcc tca ggg 2194
Ala His Ser Lys Arg Asp Thr Lys Cys Gln Pro Gln Gly Ser Ser Gly
660 665 670
gag gag aag ggg acc ccg acc aca ctc agg ggt gga gaa gca tct gaa 2242
Glu Glu Lys Gly Thr Pro Thr Thr Leu Arg Gly Gly Glu Ala Ser Glu
675 680 685
aga aaa agg ccg gac tcg ggc tgt tca act tca aaa gac acc aag tac 2290
Arg Lys Arg Pro Asp Ser Gly Cys Ser Thr Ser Lys Asp Thr Lys Tyr
690 695 700
cag tcg gtg tac gtc ata tcc gag gag aag gat gag tgc gtc ata gca 2338
Gln Ser Val Tyr Val Ile Ser Glu Glu Lys Asp Glu Cys Val Ile Ala
705 710 715 720
act gag gtg taaaatggaa gtgagatggc aagactcccg tttctcttaa 2387
Thr Glu Val

aataagtaaa attccaagga tatatgcccc aacgaatgct gctgaagagg agggaggcct 2447
cgtggactgc tgctgagaaa ccgagttcag accgagcagg ttctcctcct gaggtcctcg 2507
acgcctgccg acagcctgtc gcggcccggc cgcctgcggc actgccttcc gtgacgtcgc 2567
cgttgcacta tggacagttg ctcttaagag aatatatatt taaatgggtg aactgaatta 2627
cgcataagaa gcatgcactg cctgagtgta tatttt 2663
<210> 9
<211> 4208
<212> DNA
<213> Homo sapiens
<400> 9
ggccggcccg cgagctaggc tggttttttt ttttctcccc tccctccccc ctttttccat 60
gcagctgatc taaaagggaa taaaaggctg cgcataatca taataataaa agaaggggag 120
cgcgagagaa ggaaagaaag ccgggaggtg gaagaggagg gggagcgtct caaagaagcg 180
atcagaataa taaaaggagg ccgggctctt tgccttctgg aacgggccgc tcttgaaagg 240
gcttttgaaa agtggtgttg ttttccagtc gtgcatgctc caatcggcgg agtatattag 300
agccgggacg cggcggccgc aggggcagcg gcgacggcag caccggcggc agcaccagcg 360
cgaacagcag cggcggcgtc ccgagtgccc gcggcgcgcg gcgcagcg atg cgt tcc 417
Met Arg Ser
1
cca cgg acg cgc ggc cgg tcc ggg cgc ccc cta agc ctc ctg ctc gcc 465
Pro Arg Thr Arg Gly Arg Ser Gly Arg Pro Leu Ser Leu Leu Leu Leu Ala
5 10 15
ctg ctc tgt gcc ctg cga gcc aag gtg tgt ggg gcc tcg ggt cag ttc 513
Leu Leu Cys Ala Leu Arg Ala Lys Val Cys Gly Ala Ser Gly Gln Phe
20 25 30 35
gag ttg gag atc ctg tcc atg cag aac gtg aac ggg gag ctg cag aac 561
Glu Leu Glu Ile Leu Ser Met Gln Asn Val Asn Gly Glu Leu Gln Asn
40 45 50
ggg aac tgc tgc ggc ggc gcc cgg aac ccg gga gac cgc aag tgc acc 609
Gly Asn Cys Cys Gly Gly Ala Arg Asn Pro Gly Asp Arg Lys Cys Thr
55 60 65
cgc gac gag tgt gac aca tac ttc aaa gtg tgc ctc aag gag tat cag 657
Arg Asp Glu Cys Asp Thr Tyr Phe Lys Val Cys Leu Lys Glu Tyr Gln
70 75 80
tcc cgc gtc acg gcc ggg ggg ccc tgc agc ttc ggc tca ggg tcc acg 705
Ser Arg Val Thr Ala Gly Gly Pro Cys Ser Phe Gly Ser Gly Ser Thr
85 90 95
cct gtc atc ggg ggc aac acc ttc aac ctc aag gcc agc cgc ggc aac 753
Pro Val Ile Gly Gly Asn Thr Phe Asn Leu Lys Ala Ser Arg Gly Asn
100 105 110 115
gac cgc aac cgc atc gtg ctg cct ttc agt ttc gcc tgg ccg agg tcc 801
Asp Arg Asn Arg Ile Val Leu Pro Phe Ser Phe Ala Trp Pro Arg Ser
120 125 130
tat acg ttg ctt gtg gag gcg tgg gat tcc agt aat gac acc gtt caa 849
Tyr Thr Leu Leu Val Glu Ala Trp Asp Ser Ser Asn Asp Thr Val Gln
135 140 145
cct gac agt att att gaa aag gct tct cac tcg ggc atg atc aac ccc 897
Pro Asp Ser Ile Ile Glu Lys Ala Ser His Ser Gly Met Ile Asn Pro
150 155 160
agc cgg cag tgg cag acg ctg aag cag aac acg ggc gtt gcc cac ttt 945
Ser Arg Gln Trp Gln Thr Leu Lys Gln Asn Thr Gly Val Ala His Phe
165 170 175
gag tat cag atc cgc gtg acc tgt gat gac tac tac tat ggc ttt ggc 993
Glu Tyr Gln Ile Arg Val Thr Cys Asp Asp Tyr Tyr Tyr Gly Phe Gly
180 185 190 195
tgc aat aag ttc tgc cgc ccc aga gat gac ttc ttt gga cac tat gcc 1041
Cys Asn Lys Phe Cys Arg Pro Arg Asp Asp Phe Phe Gly His Tyr Ala
200 205 210
tgt gac cag aat ggc aac aaa act tgc atg gaa ggc tgg atg ggc ccc 1089
Cys Asp Gln Asn Gly Asn Lys Thr Cys Met Glu Gly Trp Met Gly Pro
215 220 225
gaa tgt aac aga gct att tgc cga caa ggc tgc agt cct aag cat ggg 1137
Glu Cys Asn Arg Ala Ile Cys Arg Gln Gly Cys Ser Pro Lys His Gly
230 235 240
tct tgc aaa ctc cca ggt gac tgc agg tgc cag tac ggc tgg caa ggc 1185
Ser Cys Lys Leu Pro Gly Asp Cys Arg Cys Gln Tyr Gly Trp Gln Gly
245 250 255
ctg tac tgt gat aag tgc atc cca cac ccg gga tgc gtc cac ggc atc 1233
Leu Tyr Cys Asp Lys Cys Ile Pro His Pro Gly Cys Val His Gly Ile
260 265 270 275
tgt aat gag ccc tgg cag tgc ctc tgt gag acc aac tgg ggc ggc cag 1281
Cys Asn Glu Pro Trp Gln Cys Leu Cys Glu Thr Asn Trp Gly Gly Gln
280 285 290
ctc tgt gac aaa gat ctc aat tac tgt ggg act cat cag ccg tgt ctc 1329
Leu Cys Asp Lys Asp Leu Asn Tyr Cys Gly Thr His Gln Pro Cys Leu
295 300 305
aac ggg gga act tgt agc aac aca ggc cct gac aaa tat cag tgt tcc 1377
Asn Gly Gly Thr Cys Ser Asn Thr Gly Pro Asp Lys Tyr Gln Cys Ser
310 315 320
tgc cct gag ggg tat tca gga ccc aac tgt gaa att gct gag cac gcc 1425
Cys Pro Glu Gly Tyr Ser Gly Pro Asn Cys Glu Ile Ala Glu His Ala
325 330 335
tgc ctc tct gat ccc tgt cac aac aga ggc agc tgt aag gag acc tcc 1473
Cys Leu Ser Asp Pro Cys His Asn Arg Gly Ser Cys Lys Glu Thr Ser
340 345 350 355
ctg ggc ttt gag tgt gag tgt tcc cca ggc tgg acc ggc ccc aca tgc 1521
Leu Gly Phe Glu Cys Glu Cys Ser Pro Gly Trp Thr Gly Pro Thr Cys
360 365 370
tct aca aac att gat gac tgt tct cct aat aac tgt tcc cac ggg ggc 1569
Ser Thr Asn Ile Asp Asp Cys Ser Pro Asn Asn Cys Ser His Gly Gly
375 380 385
acc tgc cag gac ctg gtt aac gga ttt aag tgt gtg tgc ccc cca cag 1617
Thr Cys Gln Asp Leu Val Asn Gly Phe Lys Cys Val Cys Pro Pro Gln
390 395 400
tgg act ggg aaa acg tgc cag tta gat gca aat gaa tgt gag gcc aaa 1665
Trp Thr Gly Lys Thr Cys Gln Leu Asp Ala Asn Glu Cys Glu Ala Lys
405 410 415
cct tgt gta aac gcc aaa tcc tgt aag aat ctc att gcc agc tac tac 1713
Pro Cys Val Asn Ala Lys Ser Cys Lys Asn Leu Ile Ala Ser Tyr Tyr
420 425 430 435
tgc gac tgt ctt ccc ggc tgg atg ggt cag aat tgt gac ata aat att 1761
Cys Asp Cys Leu Pro Gly Trp Met Gly Gln Asn Cys Asp Ile Asn Ile
440 445 450
aat gac tgc ctt ggc cag tgt cag aat gac gcc tcc tgt cgg gat ttg 1809
Asn Asp Cys Leu Gly Gln Cys Gln Asn Asp Ala Ser Cys Arg Asp Leu
455 460 465
gtt aat ggt tat cgc tgt atc tgt cca cct ggc tat gca ggc gat cac 1857
Val Asn Gly Tyr Arg Cys Ile Cys Pro Pro Gly Tyr Ala Gly Asp His
470 475 480
tgt gag aga gac atc gat gaa tgt gcc agc aac ccc tgt ttg aat ggg 1905
Cys Glu Arg Asp Ile Asp Glu Cys Ala Ser Asn Pro Cys Leu Asn Gly
485 490 495
ggt cac tgt cag aat gaa atc aac aga ttc cag tgt ctg tgt ccc act 1953
Gly His Cys Gln Asn Glu Ile Asn Arg Phe Gln Cys Leu Cys Pro Thr
500 505 510 515
ggt ttc tct gga aac ctc tgt cag ctg gac atc gat tat tgt gag cct 2001
Gly Phe Ser Gly Asn Leu Cys Gln Leu Asp Ile Asp Tyr Cys Glu Pro
520 525 530
aat ccc tgc cag aac ggt gcc cag tgc tac aac cgt gcc agt gac tat 2049
Asn Pro Cys Gln Asn Gly Ala Gln Cys Tyr Asn Arg Ala Ser Asp Tyr
535 540 545
ttc tgc aag tgc ccc gag gac tat gag ggc aag aac tgc tca cac ctg 2097
Phe Cys Lys Cys Pro Glu Asp Tyr Glu Gly Lys Asn Cys Ser His Leu
550 555 560
aaa gac cac tgc cgc acg acc ccc tgt gaa gtg att gac agc tgc aca 2145
Lys Asp His Cys Arg Thr Thr Pro Cys Glu Val Ile Asp Ser Cys Thr
565 570 575
gtg gcc atg gct tcc aac gac aca cct gaa ggg gtg cgg tat att tcc 2193
Val Ala Met Ala Ser Asn Asp Thr Pro Glu Gly Val Arg Tyr Ile Ser
580 585 590 595
tcc aac gtc tgt ggt cct cac ggg aag tgc aag agt cag tcg gga ggc 2241
Ser Asn Val Cys Gly Pro His Gly Lys Cys Lys Ser Gln Ser Gly Gly
600 605 610
aaa ttc acc tgt gac tgt aac aaa ggc ttc acg gga aca tac tgc cat 2289
Lys Phe Thr Cys Asp Cys Asn Lys Gly Phe Thr Gly Thr Tyr Cys His
615 620 625
gaa aat att aat gac tgt gag agc aac cct tgt aga aac ggt ggc act 2337
Glu Asn Ile Asn Asp Cys Glu Ser Asn Pro Cys Arg Asn Gly Gly Thr
630 635 640
tgc atc gat ggt gtc aac tcc tac aag tgc atc tgt agt gac ggc tgg 2385
Cys Ile Asp Gly Val Asn Ser Tyr Lys Cys Ile Cys Ser Asp Gly Trp
645 650 655
gag ggg gcc tac tgt gaa acc aat att aat gac tgc agc cag aac ccc 2433
Glu Gly Ala Tyr Cys Glu Thr Asn Ile Asn Asp Cys Ser Gln Asn Pro
660 665 670 675
tgc cac aat ggg ggc acg tgt cgc gac ctg gtc aat gac ttc tac tgt 2481
Cys His Asn Gly Gly Thr Cys Arg Asp Leu Val Asn Asp Phe Tyr Cys
680 685 690
gac tgt aaa aat ggg tgg aaa gga aag acc tgc cac tca cgt gac agt 2529
Asp Cys Lys Asn Gly Trp Lys Gly Lys Thr Cys His Ser Arg Asp Ser
695 700 705
cag tgt gat gag gcc acg tgc aac aac ggt ggc acc tgc tat gat gag 2577
Gln Cys Asp Glu Ala Thr Cys Asn Asn Gly Gly Thr Cys Tyr Asp Glu
710 715 720
ggg gat gct ttt aag tgc atg tgt cct ggc ggc tgg gaa gga aca acc 2625
Gly Asp Ala Phe Lys Cys Met Cys Pro Gly Gly Trp Glu Gly Thr Thr
725 730 735
tgt aac ata gcc cga aac agt agc tgc ctg ccc aac ccc tgc cat aat 2673
Cys Asn Ile Ala Arg Asn Ser Ser Cys Leu Pro Asn Pro Cys His Asn
740 745 750 755
ggg ggc aca tgt gtg gtc aac ggc gag tcc ttt acg tgc gtc tgc aag 2721
Gly Gly Thr Cys Val Val Asn Gly Glu Ser Phe Thr Cys Val Cys Lys
760 765 770
gaa ggc tgg gag ggg ccc atc tgt gct cag aat acc aat gac tgc agc 2769
Glu Gly Trp Glu Gly Pro Ile Cys Ala Gln Asn Thr Asn Asp Cys Ser
775 780 785
cct cat ccc tgt tac aac agc ggc acc tgt gtg gat gga gac aac tgg 2817
Pro His Pro Cys Tyr Asn Ser Gly Thr Cys Val Asp Gly Asp Asn Trp
790 795 800
tac cgg tgc gaa tgt gcc ccg ggt ttt gct ggg ccc gac tgc aga ata 2865
Tyr Arg Cys Glu Cys Ala Pro Gly Phe Ala Gly Pro Asp Cys Arg Ile
805 810 815
aac atc aat gaa tgc cag tct tca cct tgt gcc ttt gga gcg acc tgt 2913
Asn Ile Asn Glu Cys Gln Ser Ser Pro Cys Ala Phe Gly Ala Thr Cys
820 825 830 835
gtg gat gag atc aat ggc tac cgg tgt gtc tgc cct cca ggg cac agt 2961
Val Asp Glu Ile Asn Gly Tyr Arg Cys Val Cys Pro Pro Gly His Ser
840 845 850
ggt gcc aag tgc cag gaa gtt tca ggg aga cct tgc atc acc atg ggg 3009
Gly Ala Lys Cys Gln Glu Val Ser Gly Arg Pro Cys Ile Thr Met Gly
855 860 865
agt gtg ata cca gat ggg gcc aaa tgg gat gat gac tgt aat acc tgc 3057
Ser Val Ile Pro Asp Gly Ala Lys Trp Asp Asp Asp Cys Asn Thr Cys
870 875 880
cag tgc ctg aat gga cgg atc gcc tgc tca aag gtc tgg tgt ggc cct 3105
Gln Cys Leu Asn Gly Arg Ile Ala Cys Ser Lys Val Trp Cys Gly Pro
885 890 895
cga cct tgc ctg ctc cac aaa ggg cac agc gag tgc ccc agc ggg cag 3153
Arg Pro Cys Leu Leu His Lys Gly His Ser Glu Cys Pro Ser Gly Gln
900 905 910 915
agc tgc atc ccc atc ctg gac gac cag tgc ttc gtc cac ccc tgc act 3201
Ser Cys Ile Pro Ile Leu Asp Asp Gln Cys Phe Val His Pro Cys Thr
920 925 930
ggt gtg ggc gag tgt cgg tct tcc agt ctc cag ccg gtg aag aca aag 3249
Gly Val Gly Glu Cys Arg Ser Ser Ser Leu Gln Pro Val Lys Thr Lys
935 940 945
tgc acc tct gac tcc tat tac cag gat aac tgt gcg aac atc aca ttt 3297
Cys Thr Ser Asp Ser Tyr Tyr Gln Asp Asn Cys Ala Asn Ile Thr Phe
950 955 960
acc ttt aac aag gag atg atg tca cca ggt ctt act acg gag cac att 3345
Thr Phe Asn Lys Glu Met Met Ser Pro Gly Leu Thr Thr Glu His Ile
965 970 975
tgc agt gaa ttg agg aat ttg aat att ttg aag aat gtt tcc gct gaa 3393
Cys Ser Glu Leu Arg Asn Leu Asn Ile Leu Lys Asn Val Ser Ala Glu
980 985 990 995
tat tca atc tac atc gct tgc gag cct tcc cct tca gcg aac aat gaa 3441
Tyr Ser Ile Tyr Ile Ala Cys Glu Pro Ser Pro Ser Ala Asn Asn Glu
1000 1005 1010
ata cat gtg gcc att tct gct gaa gat ata cgg gat gat ggg aac ccg 3489
Ile His Val Ala Ile Ser Ala Glu Asp Ile Arg Asp Asp Gly Asn Pro
1015 1020 1025
atc aag gaa atc act gac aaa ata atc gat ctt gtt agt aaa cgt gat 3537
Ile Lys Glu Ile Thr Asp Lys Ile Ile Asp Leu Val Ser Lys Arg Asp
1030 1035 1040
gga aac agc tcg ctg att gct gcc gtt gca gaa gta aga gtt cag agg 3585
Gly Asn Ser Ser Leu Ile Ala Ala Val Ala Glu Val Arg Val Gln Arg
1045 1050 1055
cgg cct ctg aag aac aga aca gat ttc ctt gtt ccc ttg ctg agc tct 3633
Arg Pro Leu Lys Asn Arg Thr Asp Phe Leu Val Pro Leu Leu Ser Ser
1060 1065 1070 1075
gtc tta act gtg gct tgg atc tgt tgc ttg gtg acg gcc ttc tac tgg 3681
Val Leu Thr Val Ala Trp Ile Cys Cys Leu Val Thr Ala Phe Tyr Trp
1080 1085 1090
tgc ctg cgg aag cgg cgg aag ccg ggc agc cac aca cac tca gcc tct 3729
Cys Leu Arg Lys Arg Arg Lys Pro Gly Ser His Thr His Ser Ala Ser
1095 1100 1105
gag gac aac acc acc aac aac gtg cgg gag cag ctg aac cag atc aaa 3777
Glu Asp Asn Thr Thr Asn Asn Val Arg Glu Gln Leu Asn Gln Ile Lys
1110 1115 1120
aac ccc att gag aaa cat ggg gcc aac acg gtc ccc atc aag gat tat 3825
Asn Pro Ile Glu Lys His Gly Ala Asn Thr Val Pro Ile Lys Asp Tyr
1125 1130 1135
gag aac aag aac tcc aaa atg tct aaa ata agg aca cac aat tct gaa 3873
Glu Asn Lys Asn Ser Lys Met Ser Lys Ile Arg Thr His Asn Ser Glu
1140 1145 1150 1155
gta gaa gag gac gac atg gac aaa cac cag cag aaa gcc cgg ttt gcc 3921
Val Glu Glu Asp Asp Met Asp Lys His Gln Gln Lys Ala Arg Phe Ala
1160 1165 1170
aag cag ccg gcg tac acg ctg gta gac aga gaa gag aag ccc ccc aac 3969
Lys Gln Pro Ala Tyr Thr Leu Val Asp Arg Glu Glu Lys Pro Pro Asn
1175 1180 1185
ggc acg ccg aca aaa cac cca aac tgg aca aac aaa cag gac aac aga 4017
Gly Thr Pro Thr Lys His Pro Asn Trp Thr Asn Lys Gln Asp Asn Arg
1190 1195 1200
gac ttg gaa agt gcc cag agc tta aac cga atg gag tac atc gta 4062
Asp Leu Glu Ser Ala Gln Ser Leu Asn Arg Met Glu Tyr Ile Val
1205 1210 1215
tagcagaccg cgggcactgc cgccgctagg tagagtctga gggcttgtag ttctttaaac 4122
tgtcgtgtca tactcgagtc tgaggccgtt gctgacttag aatccctgtg ttaatttaag 4182
ttttgacaag ctggcttaca ctggca 4208
<210> 10
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 10
gat tat aaa gat gat gat gat aaa tga 27
Asp Tyr Lys Asp Asp Asp Asp Lys
1 5
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 11
tggcartgya aytgycarga 20
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 12
atyttyttyt crcarttraa 20
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 13
tgcststgyg anaccaactg 20
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 14
tttatktcrc awktckgwcc 20
<210> 15
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 15
tcgcgcgtgg agcgaagcag catgg 25
<210> 16
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 16
ggaattcgat atcaagctta tcgat 25
<210> 17
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 17
tcacccgccc tggccctcta gcttctca 28
<210> 18
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 18
ggacgcgtgg atccactagt tctagagc 28
<210> 19
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 19
tcatttatca tcatcatctt tataatcccc gccctggccc tctagcttct cagtg 55
<210> 20
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 20
aaggatcccg agggtgtctg ctggaagcca ggctca 36
<210> 21
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 21
cctctagagt cgcggccgtc gcactcattt acc 33
<210> 22
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 22
aaggatcccc gccctggccc tctagcttc 29
<210> 23
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 23
cctctagacg cgtagagcgg ccgccaccgc ggtgga 36
<210> 24
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 24
tcacacctca gttgctatga cgcac 25
<210> 25
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 25
ggacgcgtgg atccactagt tctagagc 28
<210> 26
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 26
tcatttatca tcatcatctt tataatccac ctcagttgct atgacgcact c 51
<210> 27
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 27
cggcgcagcg atgcgttccc cacgg 25
<210> 28
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 28
ggaattcgat atcaagctta tcgat 25
<210> 29
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 29
tcaatctgtt ctgttgttca gaggccg 27
<210> 30
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 30
ggacgcgtgg atccactagt tctagagc 28
<210> 31
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 31
tcatttatca tcatcatctt tataatcatc tgttctgttg ttcagaggcc g 51
<210> 32
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 32
aaggatccgt tctgttgttc agaggccgcc t 31
<210> 33
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 33
cctctagacg cgtagagcgg ccgccaccgc ggtgga 36
<210> 34
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 34
ctatacgatg tactccattc ggtttaag 28
<210> 35
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 35
ggacgcgtct agagtcgacc tgcaggcatg c 31
<210> 36
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 36
ctatttatca tcatcatctt tataatctac gatgtactcc attcggttta ag 52

Claims (18)

A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 5 in the sequence listing. The polypeptide of Claim 1 containing the amino acid sequence set forth in SEQ ID NO: 6 in the Sequence Listing. The polypeptide of Claim 1 containing the amino acid sequence set forth in SEQ ID NO: 7 in the Sequence Listing. The polypeptide according to claims 1 to 3, comprising the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing. A method for inhibiting differentiation of undifferentiated cells in vitro using the polypeptide according to claim 1. The method according to claim 5, wherein the undifferentiated cells are undifferentiated cells other than cranial nerves and muscle undifferentiated cells. The method according to claim 5, wherein the undifferentiated cells are blood undifferentiated cells. A differentiation inhibitor containing the polypeptide according to claim 1. A cell culture medium containing the polypeptide according to claim 1. The cell culture medium according to claim 9, wherein the cells are blood undifferentiated cells. A DNA encoding the polypeptide according to claim 1. The DNA according to claim 11, comprising the DNA sequence of No. 502 to No. 1095 described in SEQ ID No. 9 of the Sequence Listing. The DNA according to claim 11, comprising the DNA sequence of Nos. 502 to 3609 described in SEQ ID No. 9 of the Sequence Listing. The DNA according to claim 11, comprising the DNA sequence of Nos. 502 to 4062 described in SEQ ID No. 9 of the Sequence Listing. A recombinant DNA obtained by ligating a DNA selected from the DNA group according to claim 11 and a vector DNA that can be expressed in a host cell. A cell transformed with the recombinant DNA according to claim 15. The method for producing a polypeptide according to any one of claims 1 to 4, wherein the cell according to claim 16 is cultured, and a compound produced from the culture is collected. An antibody that specifically recognizes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 7 in the Sequence Listing.

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