WO1995015386A1 - Novel receptor tyrosine kinase - Google Patents

Abstract

A novel receptor tyrosine kinase which expresses in a nondifferentiated blood cell but undergoes a reduction in the expression level with the progress of differentiation of the nondifferentiated cell; and an antibody reactive with the above kinase. The use of this antibody makes it possible to isolate a nondifferentiated blood cell containing the above kinase bound to its surface from a biological sample containing such a cell and to detect specifically such a cell contained in a biological sample. Also disclosed is a method of screening a chemical substance which at least inhibits or activates the receptor tyrosine kinase activity or suppresses the expression of the kinase. Further disclosed are a DNA coding for the above kinase; a replicable recombinant DNA prepared by integrating expressibly the above DNA into a replicable expression vector; a microorganism or eucaryotic cell transformed by the above recombinant DNA; a sense DNA fragment, an antisense DNA fragment, and derivatives thereof, each prepared by utilizing a cDNA coding for the above kinase; and a sense RNA fragment, an antisense RNA fragment, and derivatives thereof.

Description

 Title of invention

 Novel receptor tyrosine kinase

 Background of the Invention

Technical field

The present invention relates to a novel receptor tyrosine kinase, and an antibody reactive with the receptor tyrosine kinase. More specifically, the present invention relates to a novel receptor tyrosine kinase, which is expressed in undifferentiated blood cells, but whose expression level decreases with the differentiation of undifferentiated blood cells, and the receptor tyrosine kinase. The present invention relates to an antibody reactive with a kinase. By using an antibody reactive with the receptor tyrosine kinase of the present invention, the blood sample containing the receptor tyrosine kinase on the surface contained in a biological sample can be obtained. Differentiated cells can be isolated from the biological sample, and the above-mentioned undifferentiated blood cells contained in the biological sample can be specifically detected. Furthermore, the present invention also relates to a method for screening a chemical substance capable of inhibiting or activating at least its receptor tyrosine kinase activity or inhibiting its expression. Further, the present invention provides a DNA encoding the receptor type 1 tyrosine kinase; a DNA encoding the receptor type tyrosine kinase described above, which can be expressed in a replicable expression vector. A replicable recombinant DNA; and a microorganism or eukaryotic cell transformed with the recombinant DNA; Sense DNA fragments and antisense DNA fragments prepared using cDNA encoding the above receptor type 1 tyrosine kinase, and derivatives thereof; and sense RNA fragments, antisense RNA fragments, and the like. Related to derivatives.

Conventional technology

 There are many types of cells in human blood, each of which plays an important role. For example, red blood cells carry oxygen in the body, platelets provide a haemostatic effect, and white blood cells make up the immune system to protect against infection. These diverse cells are derived from hematopoietic stem cells in the bone marrow. It has recently been shown that hematopoietic stem cells are affected by various hematopoietic and environmental factors in the body and differentiate into various blood cells, osteoclasts, mast cells, and the like. Erythropoietin (EPO) for differentiation into erythrocytes and granulocyte colony-stimulating factor (G-CSF) for differentiation into leukocytes have been discovered as hematopoietic factors. ing. However, there are still many unclear points about the mechanism of differentiation and proliferation of blood undifferentiated cells such as bone marrow cells including hematopoietic stem cells, peripheral blood cells, and umbilical cord blood cells into blood cells. In addition, some factors that differentiate and proliferate this cell group have not been found despite the fact that their existence has been pointed out, and an effective method for discovering such factors has also been found. Not.

Recent studies have revealed that tyrosine kinases are significantly involved in the development and differentiation of animals and insects. It is considered that tyrosine kinase is also greatly involved in the differentiation of differentiated cells.

 Tyrosine kinase, an enzyme that specifically phosphorylates the amino acid ticosin present in proteins, regulates signal transduction from the outside of the cell to the cell, and regulates gene transcription in the cell nucleus. It is an important substance. It is also known that translocations and point mutations of genes encoding these tyrosine kinases cause abnormalities such as canceration of cells. Furthermore, it is known that infection with a virus having a gene similar to the gene encoding these causes cancer of cells and cancer of living organisms. Therefore, understanding tyrosine kinases and their gene structure and protein structure are extremely important for the diagnosis and treatment of diseases caused by abnormal cell proliferation such as cancer. For example, using a gene amplification method such as PCR (Polymerase Chain Reaction), DNA or RNA probes, or using restriction enzymes, the tyrosine kinase gene may Alternatively, by detecting all of them, the presence or absence of point mutations, gene recombination, and expression levels in the living body can be examined and used for diagnosing diseases such as blood diseases.

Tyrosine kinase is an enzyme that phosphorylates tyrosine residues in proteins, and its physiologically active site is composed of about 250 amino acid residues. In addition, tyrosine kinases Very conserved sequences are among the amino acid sequences

(Hanks et a 1. Science 241: 42, 1988), designed a DNA sequence corresponding to the conserved amino acid sequence, and

Transcription) — New tyrosine kinase gene fragment can be obtained when used as a primer in the PCR method.

 (Wi 1 ks. Methods in Enzymology 200: 533, 1991).

 C-kit, a type of receptor tyrosine kinase, is expressed on the surface of undifferentiated blood cells, and a monoclonal antibody against c-kit is involved in the study of isolation and proliferation of undifferentiated blood cells. It was used in research, and it was pointed out that the amount of c-kit in blood correlated with the specific pathology of leukemia, and the concentration of c-kit in blood using a monoclonal antibody was used. Measurements are being developed as diagnostics. In addition, c-kit was found to be a receptor for stem cell factor (SCF), a fertilizing cell growth factor and one of the growth factors of blood undifferentiated cells. Cell 63: 5, 1992), and cell differentiation is controlled through this receptor tyrosine kinase. Such a ligand that specifically binds to the extracellular portion of the receptor type 1 tyrosine kinase generally increases the enzymatic activity in the cell upon binding, and transmits information to the cell. Generate bioactivity.

 Summary of the Invention

An effective tool for studying the differentiation process of blood cells is To use a cell line or cells that meet the purpose of this study, a human megakaryoblastic leukemia cell line UT_7 (obtained from Lecturer, Department of Hematology, Jichi Medical University, Jichi Medical University, Japan) Is a cell line that proliferates depending on hematopoietic factors such as granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin 3 (IL-3). In the presence of GM-CSF, Differentiation into megakaryocytes when the car ij intensifies in PMA (Phorbol 1 2 -Myristate 13-Acetate), and erythroblasts when stimulated with butyric acid in the presence of erythropoietin (Komat su et al. Cancer Res. 51: 341, 1991). Therefore, the use of UT-7 cells is considered to be effective for studying the mechanism of differentiation of undifferentiated blood cells.

The present inventors have cloned a thymic synkinase gene involved in the differentiation of undifferentiated blood cells from human megakaryoblastic leukemia cell line UT-7 using the RT-PCR method. As a result, a novel receptor-type tyrosine kinase was found in which the expression of mRΝΝ was strongly observed in the undifferentiated state of UΤ-7 cells, but the expression was not observed with the differentiation into megakaryocytes. . Using this gene fragment as a probe, cDNA encoding the full length was obtained from a cDNA library of human placenta and human fetal liver, the entire nucleotide sequence was determined, and a novel receptor was obtained. Cells expressing the type tyrosine kinase polypeptide were prepared, and the polypeptide was further isolated. Furthermore, the reactivity with the polypeptide is A method for detecting or isolating blood undifferentiated cells using the antibody was established. Furthermore, a new method for screening chemical substances using the above-mentioned polypeptides has been established.

 Therefore, one object of the present invention is to provide a novel receptor tyrosine kinase specifically expressed in undifferentiated blood cells and a DNA encoding the same.

 Still another object of the present invention is to provide a method for reacting to the above-described receptor type 1 lipoprotein kinase, which can be used for elucidating the mechanism of differentiation and proliferation of undifferentiated blood cells. An object of the present invention is to provide an antibody having the property, and a method for isolating or detecting undifferentiated blood cells expressing the receptor tyrosine kinase polypeptide using the antibody.

 Still another object of the present invention is to provide a receptor-type tyrosine kinase of the present invention, which has the ability to inhibit or activate receptor tyrosine kinase activity or the ability to suppress the expression of the receptor tyrosine kinase. An object of the present invention is to provide a method for screening a chemical substance such as blood undifferentiated cell differentiation / growth factor, which can regulate tyrosine kinase signal transduction.

Still another object of the present invention is to provide a sense DNA fragment and an antisense, which can be advantageously used for confirming expression of the receptor-type oral synthase gene of the present invention and for regulating gene expression in cells. DNA fragments and their derivatives, An object of the present invention is to provide a sense RNA fragment, an antisense RNA fragment and a derivative thereof.

 The above and other objects, features and advantages of the present invention will become apparent from the following detailed description and the appended claims, with reference to the accompanying Sequence Listing.

 Brief description of the sequence listing

 In the sequence listing:

 The amino acid sequence of SEQ ID NO: 1 is the sequence of the extracellular domain of the receptor tyrosine kinase of the present invention excluding the signal peptide, and the amino acid sequence of the present invention shown in SEQ ID NO: 4 Corresponding to the amino acid numbers 1 to 522 of the entire amino acid sequence of the scepter-type synthase;

The amino acid sequence of SEQ ID NO: 2 is the sequence of the receptor tyrosine kinase enzyme active portion contained in the intracellular domain of the receptor tyrosine kinase of the present invention, and is shown in SEQ ID NO: 4. The amino acid sequence of SEQ ID NO: 3 corresponds to the amino acid number of the amino acid sequence of the receptor tyrosine kinase of the present invention. SEQ ID NO: 4 shows the sequence of all domains of the extracellular domain, the transmembrane domain, and the intracellular domain, excluding the signal peptide, of the receptor tyrosine kinase of the present invention. Corresponding to the amino acid numbers 1 to 972 of the amino acid sequence of the receptor tyrosine kinase shown in the present invention; SEQ ID NO: 4 is the entire amino acid sequence of the receptor tyrosine kinase of the present invention, and the entire cDNA sequence of the receptor tyrosine kinase;

 SEQ ID NO: 5 is the nucleotide sequence of one of the primers used in Example 2 described below;

 SEQ ID NO: 6 is the base sequence of the antisense primer used in Example 2 described below;

 SEQ ID NO: 7 is a nucleotide sequence used in Example 8 described later and an oligopeptide encoded by the nucleotide sequence.

 The left and right ends of each amino acid sequence represented by SEQ ID NOs: 1 to 4 and 7 are the N-terminal and C-terminal, respectively, and the base sequence represented by SEQ ID NOs: 4 to 7 Are the 5 'end and the 3' end, respectively.

 Detailed description of the invention

 That is, according to one aspect of the present invention, an isolated amino acid sequence having an receptor tyrosine kinase activity and containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3 in the sequence listing is provided. Or a homologous variant of the polypeptide having a receptor tyrosine kinase activity.

According to another aspect of the present invention, an isolated DNA encoding the above-described polypeptide or homologous mutant is incorporated into the above-mentioned DNA vector replicable expression vector so as to be expressible. Replicable recombinant DNA, characterized by the replicable recombinant DNA A transformed microorganism or eukaryotic cell is provided.

 In the present invention, the term “receptor tyrosine kinase activity” refers to an enzyme activity that tyrosine kinase originally has to phosphorylate tyrosine residues, and the extracellular domain of receptor tyrosine kinase. It recognizes and binds ligands, and phosphorylates amino acid residues (mainly tyrosine residues) in the intracellular domain of receptor tyrosine kinases. At least one of the functions of binding to another intracellular protein at the phosphorylated site and phosphorylating the intracellular protein at the binding site is included.

 The receptor tyrosine kinase of the present invention contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3 in the Sequence Listing, but is not a species known to occur in nature. Homologous mutants having receptor tyrosine kinase activity, which are generated by mutations such as mutations, Included in kinases.

In addition, even if there is no mutation at the amino acid level, the degeneracy of the genetic code changes the amino acid sequence encoded by the DNA in chromosomal DNA or cDNA isolated from nature. There are often cases in which the DNA base sequence has been mutated without incident. Since the 5 'untranslated region and the 3' untranslated region do not participate in the definition of the amino acid sequence of the polypeptide, DNA sequences are easily mutated. Nucleotide sequences obtained by such degeneracy of the genetic code are also included in the DNA of the present invention.

 Further, a polypeptide having the receptor tyrosine kinase activity described above and having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3 has a receptor tyrosine kinase activity. DNA encoding a homologous mutant is also included in the DNA of the present invention.

 In the present invention, preparation of cDNA necessary for genetic manipulation, examination of expression by Northern blot, screening by hybridization, preparation of recombinant DNA, nucleotide sequence of DNA A series of molecular biology experiments, such as determination and preparation of a cDNA library, can be performed by the method described in an ordinary experiment manual. For example, the usual test book mentioned above is, for example,

Edited by Ma niatis et al., Moe ecu lar Cl on ing, A 1 aborartory manua l, 1989, Eds., Samb rook, J., Fr its ch, EF, and Ma niatis, T., Cold Sp r ing Harbor L oboratory Presses.

The step of obtaining the cDNA of the receptor tyrosine kinase of the present invention is as described in Examples 1 to 6 described below. That is, first, PCR is performed using a primer corresponding to an amino acid sequence characteristic of a known tyrosine kinase gene. Preparation of primers for PCR and PCR can be performed by the method of Wi 1 ks (Proc. Nat 1. Acad. Sc. USA 86: 1603, 1989). You That is, an appropriate oligonucleotide is prepared using a commercially available DNA synthesizer and purified to obtain a primer for PCR. Next, PCR primers were added to the above-described cDNA solution of the human megakaryoblastic leukemia cell line UT-7, and PCR was performed.The resulting PCR product was cloned and the gene sequence was determined. It corresponded to the nucleotide sequence of SEQ ID NO: 4 from No. 2642 to No. 2812. This sequence encodes the central portion of the tyrosine kinase enzyme active site and encodes an amino acid sequence that plays an important role in intracellular signaling.

 To clone the entire sequence of the receptor tyrosine kinase cDNA of the present invention, the cDNA fragment cloned by the above method is labeled with an isotope or a non-isotope, and the UT-17 cell line is cloned. c DNA libraries can be obtained by screening by a method such as hybridization. For example, the labeling method for isotope is

[ ³² P] y — The method of labeling the ends using ATP and T4 polynucleotide kinase, or other labeling methods such as the nick translation method or the primer extension method can be used.

The DNA sequence of the receptor tyrosine kinase of the present invention thus obtained is shown in the sequence listing as SEQ ID NO: 4 together with the amino acid sequence encoded by it. The base sequence is a 5 'untranslated region consisting of 409 bases, followed by a region coding for the receptor tyrosine kinase of the present invention consisting of 296 bases, It consists of a 3 'untranslated region consisting of 919 bases. Further, the amino acid sequence of the receptor type 1 tyrosine kinase of the present invention comprises 15 amino acids corresponding to amino acids 15 to 11 of SEQ ID NO: 4 in the sequence listing. Signal peptide, the extracellular portion consisting of amino acids 2 to 5 in the amino acid sequence of SEQ ID NO: 4 in SEQ ID NO: 4 and SEQ ID NO: 4 in the SEQ ID NO: 4 A transmembrane portion composed of 26 amino acids, corresponding to amino acids Nos. 5 2 3 to 5 4 8 in the amino acid sequence, 5 4 9 to 9 9 in the amino acid sequence of SEQ ID NO: 4 in the sequence listing 72 The intracellular portion composed of 4 2 4 amino acid, which is the second component, is composed of the same. Furthermore, in the intracellular portion, 260 amino acid, which corresponds to amino acids 600 to 859 of the amino acid sequence of SEQ ID NO: 4 in the sequence listing, is a tyrosine kinase enzyme active portion. The transformed cell obtained by transfecting the vector pBSRTKFULL containing the entire nucleotide sequence of the receptor tyrosine kinase cDNA of the present invention into Escherichia coli JM109 (manufactured by Toyobo, Japan) was used. It was deposited at the Research Institute of Biotechnology, Industrial Technology Institute of the Ministry of International Trade and Industry of Japan under the deposit number FERMBP—4883 on January 11, 1974.

The cDNA of the receptor tyrosine kinase of the present invention may be a human chronic myelogenous leukemia cell line K5662 (Nippon RIKEN, Cell Development Bank) other than UT-7. RCB 0 2 7) and human acute megakaryoblastic white Hematopoietic cell line CMK (Blood 74:42, 1989) and other non-blood cell lines include the hepatocellular carcinoma cell line Hep3B [American Type Culture 'Collection (hereinafter ATCC). Available), HB8064] and human fetal lung fibroblast cell line MRC-5 (available from Nippon RIKEN, Cell Development Bank, No RCB0211) Thus, it can be obtained by substantially the same operation as in Examples 1 to 6 described later.

 The polypeptide having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing is the amino acid sequence of the receptor-type tyrosine kinase of the present invention except for the signal peptide of the extracellular portion. The polypeptide having an amino acid sequence represented by SEQ ID NO: 2 in the sequence listing corresponds to the polypeptide having the amino acid sequence of the receptor type cytosine kinase of the present invention. It corresponds to a polypeptide having an amino acid sequence in the active part. Further, the polypeptide having the amino acid sequence represented by SEQ ID NO: 3 in the sequence listing has the amino acid sequence of the portion excluding the signal peptide of the receptor tyrosine kinase of the present invention. Corresponds to a polypeptide.

The receptor tyrosine kinase peptides of the present invention are human EPH (Hirai et al., Science, 238, 1717-1720, 1987), which is a cloned receptor tyrosine kinase. , Human ECK (Lindberg and Hunter, Mo 1. Cell l. Bio 1., 10, 6316-6324, 1990), rat ELK (Lhotak et a 1- Biol., 11, 2496—2502, 1991), human HEK (Wicks et al., Proc. Nat 1. Acad. Sci. USA., 89, 1611-1615. , 1992), mouse SEK (Gi 1 ardi-He benstreiteta, One ogene, 7, 2499-2506, 1992), chicken C ek — 5

 (Pasquale, Cell Regulation, 2, 523-534, 1991), but the homologue of its amino acid sequence is 56.3% at the highest ELK. However, it has a novel amino acid sequence completely different from these genes.

 The receptor type 1 tyrosine kinase of the present invention has receptor tyrosine kinase activity by a known genetic engineering technique, and is selected from the group consisting of SEQ ID NOs: 1, 2 and 3 in the sequence listing. An expression vector capable of replicating a DNA encoding a polypeptide containing an amino acid sequence or a DNA encoding a homologous mutant of the polypeptide having a receptor tyrosine kinase activity. Thus, a transformant can be obtained by incorporating a microorganism or a eukaryotic cell (eg, an insect cell or an animal cell) as a host. When it is desired to produce the receptor tyrosine kinase of the present invention under more stable conditions, it is preferable to use eukaryotic cells, particularly cultured animal cells, as hosts.

The receptor-type tyrosine kinase of the present invention, the DNA encoding the receptor-type tyrosine kinase, and the transformant that produces the receptor-type tyrosine kinase are obtained by basic analysis of the receptor-type tyrosine kinase. From research, it has been used for the development of chemicals related to the medical field such as pharmaceuticals, including anticancer drugs that specifically regulate the signal transduction system via receptor type 1 tyrosine kinase, and for the use of drug designs. it can.

 As described above, the expression of the mRNA of the receptor tyrosine kinase gene of the present invention is observed when the human megakaryoblastic leukemia cell line UT-7 is undifferentiated. However, a similar phenomenon can be confirmed in other megakaryoblast cell lines, since the expression of mRNA is not observed upon differentiation into megakaryocytes.

As a method for detecting the expression of the receptor tyrosine kinase of the present invention in undifferentiated blood cells, a part or all of the gene sequence of SEQ ID NO: 4 in the sequence listing was designed as type III. Northern blots using synthetic oligonucleotides are also possible, but a more convenient method is to use a method using an antibody reactive with the receptor tyrosine kinase of the present invention. ,. Monoclonal and polyclonal antibodies can be prepared by a conventional method using part or all of the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing as an immunogen. In particular, an antibody against the extracellular portion (SEQ ID NO: 1) is a very effective means that can detect cells in a living state. Furthermore, as described below, the present receptor tyrosine kinase is expressed using an antibody reactive with the receptor tyrosine kinase of the present invention. Blood undifferentiated cells can also be efficiently isolated.

 That is, according to still another embodiment of the present invention, an amino acid sequence having receptor tyrosine kinase activity and selected from the group consisting of SEQ ID NOs: 1, 2, and 3 in the sequence listing is provided. The present invention provides an antibody having reactivity with a contained polypeptide or a homologous mutant of the polypeptide having receptor type 1 tyrosine kinase activity.

An example of a method for producing an antibody specific to the receptor tyrosine kinase of the present invention is shown in Example 8 described later. The three strains of hybridomas producing monoclonal antibodies produced in this way, 38--1E, 66--3A and 68--3A, were developed by the Institute of Biotechnology and Industrial Technology, the Ministry of Economy, Trade and Industry of Japan. At FE RM BP—4884, FE RM BP—4885, and FE RM BP—4886, respectively, on January 11, 1999, respectively. Has been deposited.

Antibodies reactive with the receptor tyrosine kinase of the present invention against the extracellular domain of the receptor tyrosine kinase can be prepared by using the polypeptide obtained as described above as an immunogen in a conventional manner. Polyclonal antibodies and monoclonal antibodies that specifically recognize the receptor tyrosine kinase of the present invention can be produced.

 From this monoclonal antibody, the gene encoding the amino acid sequence of the monoclonal antibody, in particular, the variable region of the antibody gene is separated by a genetic engineering method such as PCR to obtain a mouse, human or rat. A gene encoding a chimeric antibody recognizing the receptor tyrosine kinase of the present invention is obtained by connecting the gene encoding an antibody such as a rat to the gene encoding the antibody by using a genetic engineering technique, and the expression vector is used to obtain the gene. Then, the gene can be introduced into eukaryotic cells such as animal cells and prokaryotic cells such as bacterium to produce a chimeric antibody that recognizes the receptor-type oral synthase of the present invention. Furthermore, the shape of the antibody may be the whole antibody, but it may be expressed in the form of Fab type, which expresses only the variable region of the antibody, or in the form of a single-chain peptide using phage, etc. It is possible to do it. The antibodies of the present invention also include the above-mentioned chimeric antibodies, recombinant antibodies of Fab type, single chain peptide type and the like, and analogs thereof.

 The antibody of the present invention may be used, if desired, in a form immobilized on a solid support such as a petri dish or magnetic beads.

Antibodies prepared in this manner, especially the extracellular portion, are used as antigens. Monoclonal antibodies can identify small amounts of undifferentiated blood cells in blood cells floating in peripheral blood, bone marrow fluid, umbilical cord blood, etc. It can be used advantageously.

 That is, according to still another aspect of the present invention, there is provided a method for isolating a somatic cell containing a polypeptide having a receptor tyrosine kinase activity, comprising:

 (1) A polypeptide having an receptor tyrosine kinase activity and containing an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 and 3 in the sequence listing, or a tyrosine kinase of the polypeptide. The above-described antibody of the present invention is added to a biological sample containing a somatic cell having a homologous mutant having a monotase activity as an antigen on the surface thereof under the conditions where the antigen and the antibody form an antigen-antibody complex. And thereby obtain an antigen-antibody reaction mixture; and

 (2) isolating the above-mentioned somatic cells containing the polypeptide or the homologous mutant in the form of an antigen-antibody complex from an antigen-antibody reaction mixture;

Methods are provided that encompass this.

 Examples of the biological sample include an aqueous suspension containing undifferentiated blood cells and a sample composed of a body fluid containing undifferentiated blood cells.

Further, after the above step (2), the antibody is separated from the antigen-antibody complex, and the receptor-type antibody of the present invention in a form not complexed with the antibody is separated. Somatic cells containing oral synkinase can also be obtained.

 Methods for separating the antibody from the antigen-antibody complex include, for example, physical stimulation such as tapping, enzymatic treatment with proteases such as papine, chymopapine, and trypsin, pH of the solution, and salt concentration. There is a method to remove the antibody from the antigen-antibody complex due to changes. Alternatively, cells expressing the receptor tyrosine kinase of the present invention can be isolated by digesting the antigen or antibody with an enzyme.

 As a method for isolating blood undifferentiated cells, there is a method of staining the cells with the monoclonal antibody and isolating the cells by a flow cytometer. A method such as a panning method using an antibody immobilized on a substrate surface such as a shear, a column method using magnetic beads, or the like is preferable, but is not limited to these methods. Absent.

 The above method is useful not only for elucidating the mechanism of differentiation and proliferation of undifferentiated blood cells, but also for hematopoietic stem cells expressing the receptor tyrosine kinase of the present invention. The isolated blood undifferentiated cells obtained as described above have a colony formation equivalent to or higher than that of the blood undifferentiated cells isolated using the anti-CD34 antibody, as shown in Example 9 described later. It can be applied to bone marrow transplantation, peripheral blood stem cell transplantation, umbilical cord blood stem cell transplantation, and the like, which are currently performed.

According to still another aspect of the present invention, a receptor-type tyro As a method for detecting a polypeptide having a synkinase activity,

 (1) A polypeptide having an receptor tyrosine kinase activity and containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2 and 3 in the sequence listing, or a polypeptide of the same. Contacting the above-described antibody of the present invention with a biological sample containing a homologous mutant having tyrosin kinase activity as an antigen under conditions where the antigen and the antibody form an antigen-antibody complex; Thereby, an antigen-antibody reaction mixture is obtained; and

 (2) detecting the polypeptide or the homologous mutant in the form of an antigen-antibody complex,

Methods are provided that encompass this.

 Examples of the biological sample include an aqueous suspension containing undifferentiated blood cells and a sample made of a body fluid containing undifferentiated blood cells, as in the method for isolating somatic cells.

As the above detection method, a method using a flow site meter is generally used, but other known detection methods utilizing an antigen-antibody reaction can also be used. In addition, the detection of the receptor tyrosine kinase of the present invention present in serum and plasma indicates that receptor tyrosine kinases c-erb B2 and c-kit are effective as one tumor marker. Reports indicate that it could be used to detect tumor markers as well. In such a case, the antigen recognition sites of the receptor tyrosine kinase of the present invention are different. Sandwich-type enzyme antibody method using various kinds of antibodies is considered to be desirable.

 Further, according to still another aspect of the present invention, a polypeptide capable of inhibiting or activating receptor tyrosine kinase activity or having a receptor tyrosine kinase activity As a method of screening chemical substances that suppress the onset of

 (1) An isolated polypeptide having a receptor tyrosine kinase activity and containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3; Alternatively, the polypeptide is brought into contact with a homologous mutant having a receptor tyrosine kinase activity to inhibit or activate the receptor tyrosine kinase activity of the polypeptide or the homologous mutant, Alternatively, the ability to inhibit or activate at least the receptor tyrosine kinase activity of the polypeptide or the homologous variant, using the expression suppression ability of the polypeptide or the homologous variant as an index, or Detecting a chemical substance that suppresses the expression of the polypeptide or the homologous mutant; and

 (2) A method is provided which comprises isolating a detected chemical substance from a sample material.

By this method, the present invention binds to the receptor tyrosine kinase of the present invention and suppresses or promotes the autophosphorylation of the receptor tyrosine kinase. And a compound capable of inhibiting or activating the receptor tyrosine kinase activity, or a chemical substance that suppresses the expression of the receptor tyrosine kinase of the present invention, for example, Example 10 described below. Screening of interleukin 1 (IL-1α) which is a megakaryocyte differentiation factor as shown in FIG. In addition, novel chemicals such as unknown blood undifferentiated cell differentiation and growth factor, which are expected to be useful as pharmaceuticals because they can regulate the signal transduction of the receptor tyrosine kinase of the present invention. It may be possible to screen substances.

An isolated antisense DNA fragment and antisense RNA fragment paired with a part of the base sequence represented by SEQ ID NO: 4 in the sequence listing, and they are methylated, methylphosphorylated, and deflated. Derivatives that have been converted into mine or thiophosphoate can be used for research on differentiation of blood undifferentiated cells and pharmaceuticals such as agents for controlling differentiation of blood undifferentiated cells. In addition, an isolated sense DNA fragment and an antisense DNA fragment having a part of the nucleotide sequence of SEQ ID NO: 4 in the sequence listing, or a derivative thereof, and an isolated sense RNA having a sequence corresponding thereto Expression in cells can also be regulated by fragments and antisense RNA fragments or derivatives thereof. Furthermore, using these DNA fragments, RNA fragments and their derivatives, it was possible to confirm the expression of the mRNA of the receptor type 1 tyrosine kinase of the present invention as shown in Example 7 below. it can. The isolated of the present invention The sense DNA fragment, antisense DNA fragment, sense RNA fragment, and antisense RNA fragment and their derivatives may be 12 mer or more, but may be 14 mer to 16 mer or more. I want it.

BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to these Examples. Example 1 Po1y of human megakaryoblastic leukemia cell line UT-7

(A) ⁺ RNA preparation

The culture and passage of UT-7 cells (available from Professor Toshio Suda, Division of Differentiation and Control, Institution of Genetics and Medicine, Kumamoto University School of Medicine or Norio Komatsu, Department of Hematology, Jichi Medical University, Japan) Iscove's modified Dulbecco's medium (IMDM) containing 10% fetal calf serum (FCS; hereafter, FCS used in this example are all manufactured by Filtron, Australia). ) used, human granulocyte macro off § Jiko b knee stimulating factor (h GM- CSF) was added to the power sale by a 2 ng Zm l, at C 0 ₂ b Nkyubeta scratch. 3 7 conditions ° C I went in. As unstimulated cells, cells cultured under the same conditions were used. For differentiation into megakaryocytes, dilute to a cell concentration of 2 x 10 ⁵ cells Zml and convert human granulocyte monocyte colony forming factor (hGM-CSF) to Sng Zml. In addition, phorbol 12-millistate 13-acetate (PMA) (Sigma, USA) was added to a concentration of l ng / ml, and the cells were cultured for 3 days. Cells were collected. Cells cultured under each of these conditions were collected at a cell count of 1 × 10 ⁸ cells, and Dulbecco's modified phosphate buffer containing no calcium and magnesium was used (PBS (-), manufactured by Nissi Corporation, Japan). ) Wash 3 times with, Used for RNA extraction.

 Lithium chloride Z urea method (LithiumCh1oride / Urea method: Eur.J.Biochem. 107: 303)

(1980)), all RNAs were extracted. Then, O l i g o t e x

-Polly (A) at dT30 (Takara Shuzo, Japan)

⁺ RNA was separated and purified.

Example 2 Preparation of primers specific for tyrosine kinase

 According to the method of Wi 1 ks (Proc. Nat 1.Acad. Sc i. USA 86: 1603, 1989), the sub-domain of a tyrosine kinase having an amino acid sequence relatively common to known tyrosine kinases. A mixed primer corresponding to ins 7 and 9 and linked to a recognition site for a restriction enzyme, ie, a sense primer PTKI: 5'-TTGTCGACAC (AC) G (AG) GA (CT) (CT) T (CG) GC (ACGT) GC (ACGT) (AC) G-3 ,, (27mer: added a S a1I site as a recognition site for restriction enzyme, described in SEQ ID NO: 5 in Sequence Listing) and anti- Sense primer PTKII: 3'-CT (AG) CA (CG) ACC (AT) (CG) (AG) A (AT) A CCTTAAGGT-5 '(24mer: Recognition site of restriction enzyme E The following PCR was carried out using a coRI site added thereto (described in SEQ ID NO: 6 in the sequence listing).

Synthetic oligonucleotides were prepared using a fully automatic DNA synthesizer based on the solid-phase method. As a fully automatic DNA synthesizer, Applied Biosystems Inc. 391 PCR-MATE in the United States was used. Nucleotide, 3'-nucleotide Body, solutions, and reagents were used according to the company's instructions. The specified coupling reaction is completed, and the oligonucleotide carrier from which the protecting group at the 5 'end has been removed with trichloroacetic acid is left at room temperature for 1 hour in concentrated ammonia. The oligonucleotide was released from the carrier. Next, in order to release the protecting groups of the nucleic acid and the phosphoric acid, the reaction solution containing the nucleic acid is allowed to stand in a sealed vial in a concentrated ammonia solution at 55 ° C for 14 hours or more. Was. Purification of each of the oligonucleotides from which the carrier and the protecting group were released was performed using an OPC cartridge from Applied Biosystems in the United States, and detritylated with 2% trifluoroacetic acid. did. The purified primer was dissolved in deionized water to a final concentration of 1 μg / μ1 and used for PCR.

Example 3 Synthesis of c D Ν Α

CDNA was synthesized using the Po 1 y (A) ⁺ RNA obtained in Example 1. That is, 2 g of Po 1 y (A) ⁺ RNA was dissolved in 12.3 μl of deionized water, and 10X buffer solution (500 mM KC 1, 100 mM MTris-HC 1

(H 8. 3), 1 5 mM M g C 1 2 0. 0 1% gelatin) d NTPM ixture (Japan, Takara Shuzo Co., Ltd.) 4 mu 1, antisense Supuraima one specific for the above-mentioned tyrosine kinases Ρ Τ Κ Ι I (1 μg μ 1, described in SEQ ID NO: 6 in the sequence listing) 11, avian myeloblastosis reverse enzyme (avianmyeloblastosis) virusreversetranscrip tase: Life Science: 32 U I) 0.2 l and RNase inhibitor (Boehringer, Germany: 40 4 / μ1) 0.51 After standing at 37 ° C for 75 minutes, it was left at 65 ° C for 10 minutes.

Example 4 P with primers specific for tyrosine kinases

 C R

Amplification by PCR was performed as follows. Using the cDNA solution obtained in Example 3, 10X buffer solution (500 mM KC1, 100 mM Tris—HC1 (pH 8.3), 15 mM Mg) C120.0 1% gelatin) 8 µl, dNTPMixture (Takara Shuzo, Japan) 6.4 µl, sense primer specific for tyrosine kinase described above Ρ TKI (lg / jul, 1.5 μl) and 0.2 μl of Taq DNA polymerase (Amp1iTaq, manufactured by PerkinElmer Inc., USA: 5 U_iul) (described in SEQ ID No. 5 in the column list). Finally, add deionized water to bring the total volume to 10 μΟ, and prepare a process consisting of 94 ° C for 1 minute, 37 ° C for 2 minutes, and 72 ° C for 3 minutes. As one cycle, this step was performed for 40 cycles, and finally, PCR was carried out by leaving the mixture at 72 ° C for 7 minutes. A part of this PCR product was subjected to 2% agarose gel electrophoresis, stained with ethidium bromide, and observed under ultraviolet light to confirm that about 210 bp of cDNA was amplified. Example 5 Cloning of PCR product and determination of nucleotide sequence

The entire amount of the PCR product was electrophoresed on a 2% agarose gel prepared with low-melting point agarose, stained with ethidium reagent, and a band of about 210 bp was cut out under UV irradiation. After adding the same volume of distilled water and heating at 65 ° C for 10 minutes to completely dissolve the gel, add an equal amount of TE-saturated phenol (Nippon Gene, Japan) to add 1 After centrifugation at 500 rpm for 5 minutes, the upper layer was separated, and the same separation operation was performed using a TE-saturated phenol: chloroform (1: 1) solution and then chloroform. . From the finally obtained solution, cDNA was recovered by ethanol precipitation. The collected cDNA was digested with restriction enzymes EcoRI (Takara Shuzo, Japan) and Sayl (Takara Shuzo, Japan), and then used for vector integration.

As a vector, pBluescript IIKS (manufactured by Stratagene, USA; hereinafter, referred to as pB1uescript) was used to add the restriction enzymes EcoRI and Sa1I before incorporating the cDNA. And digested. The treated vector is mixed with the cDNA at a molar ratio of 1: 5, and the mixture is treated with T4 DNA ligase (New England Biolabs, USA). c DNA was incorporated. The pB1uescript into which the cDNA was incorporated was transfected into E. coli JMl09, and seeded on a plate of L-Br0th semi-solid medium containing 50 μg / ml ampicillin. 12 hours At 37 ° C, randomly select the colonies that appeared, digest the cDNA into the restriction enzymes Ec0RI and Sa1I, It was confirmed that the 10 bp cDNA was cut out, and the nucleotide sequence of the incorporated cDNA was compared with the fluorescence sequencer of Applied Biosystems Inc. It was decided on Model 373A. As a result, a tyrosine kinase gene fragment was obtained that was cloned from unstimulated UT-7 cells, but was not cloned when differentiated into megakaryocytes. This gene fragment was from the 2nd position to the 2nd position in SEQ ID NO: 4 in the sequence listing.

Example Preparation of β cDNA library and full-length cloning of novel tyrosine kinase gene and its analysis Poly (A) ⁺ RNA of unstimulated UT-7 cells isolated and purified by the method described above A cDNA library was prepared using. The UT-7 cDNA library was prepared using the pCDM 8-vector cDNA library preparation kit (manufactured by Invitrogen, The Netherlands) according to the attached preparation method. Further, Poy (A) ⁺ RNA was purified from human placenta according to the method described above to prepare a cDNA library. The human placenta cDNA library was prepared using an LZAPII cDNA library preparation kit (Stratagene, USA) according to the attached preparation method. Next, these c DNA library co from rie Ronihai Bed Li Daizesho down also properly plaque hybridization Daize one searching 5 X 1 of clones having full length c D NA with cane emissions 0 ⁵ corresponding co Roni one Or from plaque. Appeared colonies or plaques were transferred to nylon filters (Hybond N +: Amersham, UK), and the transferred nylon filters were treated with alkali (1.5 MNaCl, 0.1 M NaCl). Leave on filter paper impregnated with 5 M NaOH for 7 minutes, and then neutralize (1.5 MNaCl, 0.5 MT ris — HC 1 (PH 7.2), ImM EDTA) (3 minutes on filter paper) and then SSPE solution (0.36 MNaCl, 0.02 M sodium phosphate (pH 7.7)., 2 After shaking for 5 minutes in a 2x solution of EDTA (mM EDTA), the plate was washed and air-dried. Thereafter, the filter was allowed to stand on a filter paper impregnated with 0.4 M NaOH for 20 minutes, shaken with a 5-fold concentration SSPE solution for 5 minutes, washed, and air-dried again. Using this filter, screening was performed with a cDNA probe labeled with ³² P radioisotope.

A cDNA probe labeled with the radioactive isotope ³² P was prepared as follows. That is, pB1ue-script containing the partial cDNA of the receptor tyrosine kinase of the present invention, Sail and EcoRI were cut out of the vector and cut from the low melting point agarose gel. Purify and recover DNA fragments did. The obtained cDNA fragment is labeled with a DNA labeling kit (Megaprime DNA labeling

system: Amersham, UK). That is, to 25 ng of DNA, add 5 μl of primer solution and deionized water to make a total volume of 33 μl, perform a boiling water bath for 5 minutes, and then perform reaction buffer solution containing dNTP 10 / ^{/ 1, α - 32 P -} d CTP 5 iu 1, and the addition of T 4 DNA polynucleotidyl click Reochi Dokinaze soluble liquid 2 mu 1, and a water bath for 10 minutes at 3 7 ° C, further followed, Sefuade' click scalar (Q uick S pin

Purification was performed using a column, and the mixture was purified by a boiling water bath for 5 minutes and then cooled on ice for 2 minutes before use.

The filter prepared by the above method was applied to a final concentration of each component of 5 times concentration of SSPE solution, 5 times concentration of Denhardt's solution, 0.5% SDS (sodium dodecyl sulfate), and lOmg / I 1 of a boiling water bath for immersing the pre hive Li Dizeshiyo down solution is re denatured salmon sperm DNA, After cormorants preparative vibration 2 hours 6 5 ° C, the probes ³² P-labeled in the manner described above It was immersed in a hybridization solution having the same composition as the pre-hybridization solution containing it, and shaken at 65 ° C for 16 hours to perform hybridization.

Next, the filter is immersed in an SSPE solution containing 0.1% SDS, shaken at 65 ° C, washed twice, and further washed with 0.1% SD. It was immersed in a 10-fold diluted SSPE solution containing S and washed four times at 65 ° C. After the washing, the filters were subjected to auto-geography using an intensifying screen. As a result, the clones in the strongly exposed areas were picked up, the colonies and the blacks were sown again, and the screening was performed in the manner described above, thereby completely separating the single clones.

 The two clones with large insert sizes of the clones isolated from the UT-7 library are listed above.

Plasmid was purified according to the method described in the experimental manual of Maniatis, et al., Digested with the restriction enzyme XhoI, cDNA was purified by low-melting-point agarose electrophoresis, and incorporated into pBIuescript. The size of the incorporated cDNA was approximately 3.0 kbp and 1.6 kbp. Moreover, phage DNA was purified from clones isolated from the human placenta library, which had a large insert size, by the method described in the experimental manual by Maniatis et al. And similarly incorporated into pB1uescript. The size of the incorporated cDNA was approximately 3.8 kbp (clone 2) and 3.5 kbp (clone 9).

The gene sequences at both ends of the cDNAs of these clones were determined using the ALFDNA Sequencer from Sweden and Pharmacia ALFDNA Sequencer and the labeling kit for ALF Sequencer from Sweden and Pharmacia from Sweden, according to the attached instruction manual. did. Furthermore, in order to determine the full-length nucleotide sequence, a delivery mitigation kit for Kilo-Sequence Co., Ltd., Japan, using the kit, was prepared in accordance with the attached instruction manual. Then, the nucleotide sequences in both directions of the cDNA were determined.

 As a result, it was found that the full-length cDNA was not cloned. 1 Using a commercially available human fetus using about 200 bp of the 5 'part from the Xh0I site (corresponding to positions 484 to 696 of SEQ ID NO: 3 in the sequence listing) As a result of screening the liver cDNA library gtll (manufactured by Clontech) in the same manner, a clone containing cDNA in the 5 'direction from the Xhol site (c1oneMl, distributed) was obtained. (No. 1 to No. 124 in SEQ ID NO: 3 in the column list) were cloned, and the gene sequence was sequenced to determine the nucleotide sequence of the full-length cDNA gene of the present invention.

 The DNA having the full-length gene sequence was prepared using the DNA Xh0I site described in SEQ ID NO: 3 in the sequence listing.

By connecting c1one2 and c1oneMl, using the EcoRI site of the library linker, subcloning to the EcoRI site of pB1uescript, and finally distributing. A vector PBSRTKFULL containing a DNA fragment having the nucleotide sequence of the entire cDNA of the receptor tyrosine kinase of the present invention described in SEQ ID NO: 4 in the column list was completed. The vector containing this gene, PBSRTKFULL, was added to E. coli JM 10 9 (Toyobo, Japan) The transformed cells were deposited at the Institute of Biotechnology, Industrial Technology Research Institute, Ministry of International Trade and Industry, Ministry of International Trade and Industry of Japan under the accession number FERMBP — 4 883. Deposited on January 11th.

Example 7 Confirmation of mRNA Expression by Northern Blotting

 This is a filter to which mRNA is previously transcribed in order to examine the expression of mRNA of this novel receptor tyrosine kinase. Clone Tech, U.S.A.

M u l t i p l e T i s s u e N o r t h e r n

B l o t, H u m a n M u l t i p l e T i s s u e N o r t h e r n B l o t I I, H u m a n

F e t a l M u l t i p l e T i s s u e

Northern B 1 ot and mRNA recovered by the method described in Example 1 were subjected to agarose gel electrophoresis, and transfected into Zeta-Prob (manufactured by Biorad, USA). Using the filtered filter, PBSRTKFULL was digested with the restriction enzyme SmaI, electrophoresed on a 1% agarose gel, and a 746 bp fragment (610th position of SEQ ID NO: 4 in the sequence listing) was purified. 1 3 5 5) and then Geneclean

The gene fragment purified using I I (manufactured by BIO101, USA) was purified from the DNA labeling kit (MegaPrime kit).

DNA labelingsystem: Amasi, UK At catcher arm Ltd.) was examined ³² P-labeled expressed in the manner described above.

 As a result, expression was observed in the heart, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, prostate and ovary of adult human tissues. However, expression was not observed in brain, thymus, testis, small intestine, large intestine, or peripheral blood lymphocytes. In human fetal tissue, expression was observed in the heart, lung, liver, and kidney, and no expression was observed in the brain.

 In addition, among the blood cell lines, the myelomegakaryoblastic leukemia cell line UT-7 and the human chronic myelogenous leukemia cell line K5662 (available from RIKEN, Japan o.RCBO027) and human acute megakaryoblastic leukemia cell line CMK (Blood 74:42, 1989), and non-blood cell lines include hepatocellular carcinoma cell line Hep 3B [ American Type 'Culture. Collection (hereinafter referred to as ATCC), available from HB8064] and human fetal lung fibroblast cell line MRC-5 (RIKEN, Japan Available from the Development Bank, No. RCBO2 1 1). Furthermore, it was confirmed that expression of UT-7, K556, and CMK almost completely disappeared when cell differentiation was induced by Ρ Ρ. However, a cell line established from human cord blood 血 — 2

(Available from RIKEN, RIKEN, Cell Development Bank, No.RCB0712), Human chronic myeloid leukemia cell line KG1a

(Available from ATCC, CCL 246.1), acute No expression was observed in the leukemia cell line MOLT-4 (available from ATCC, CCL1582).

 From the above, it was inferred that this gene was expressed in an undifferentiated state of blood cells, and its expression disappeared as blood cells differentiated. Therefore, the receptor tyrosine kinase of the present invention was considered to be involved in the maintenance of blood stem cells and megakaryocyte differentiation.

Example 8 Preparation of a cell line expressing the receptor tyrosine kinase of the present invention and preparation of an antibody recognizing the receptor tyrosine kinase of the present invention

DNA coding for the polypeptide having the amino acid sequence of SEQ ID NO: 1 in the obtained sequence listing, and coding for the polypeptide having the amino acid sequence of SEQ ID NO: 2 in the sequence listing And the SV40 virus early promoter upstream of the DNA encoding the polypeptide having the amino acid sequence of SEQ ID NO: 3 in the sequence listing, and the SV01 virus P01 downstream. An expression plasmid was constructed by connecting the y (A) ⁺ signal and the dihydrofolate reductase gene. This plasmid was purified, and 20 µg thereof was introduced into CHO / dhFr- cells (ATCCCRL9906) suspended in a glucose solution. Gene transfer was carried out by applying a voltage of 600 V using Gene Pulser of Biorad, USA. The cells are cultured for two days in a medium containing 10% fetal bovine serum, and then cultured in a medium containing methotixate (MTX) agent (Dulbecco MEM, The cells were cultured in 10% fetal bovine serum) and transfected cells were selected by gene transfer. MRNA is extracted from these cells, and obtained on an oligo dT gel column.Poly (A) ⁺ RNA is obtained.After electrophoresis, it is electrically transferred to a filter. Hybridization with the receptor tyrosine kinase gene of the present invention confirmed that mRNA was expressed. As a result, it was confirmed that a transformed cell expressing the extracellular portion of the receptor tyrosine kinase of the present invention, the enzyme active portion, and the full length was obtained.

In addition, in order to confirm the expression of these proteins, amino acids of the amino acid portion from the C-terminal of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 in the sequence listing were used. A polypeptide having an acid sequence is prepared on a peptide synthesizer according to a conventional method, and the helmet is coupled with mosquisin (KLH) to produce an immunogen. The rabbit is immunized with the immunogen. Antiserum against polypeptide was obtained.

 The cell crushed product of the transformed cells and the cell culture supernatant were subjected to polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and subjected to Western blotting using the prepared antiserum. As a result, in the transformed cells expressing the extracellular portion of the receptor tyrosine kinase of the present invention, a band of about 80 kDa protein was confirmed in the cell culture supernatant. In the transformed cell lines expressing the enzymatically active portion and full length of the receptor tyrosine kinase, a protein band of 30 kDa and 130 kDa was confirmed in the cell lysate, respectively. As a result, it was confirmed that these transformed cell lines produced the corresponding polypeptides. The extracellular and full-length polypeptides are expected to be glycosylated, approximately 20 kilodaltons larger than the molecular weight expected from the amino acid composition.

Further, in order to prepare a monoclonal antibody specifically recognizing the receptor tyrosine kinase extracellular portion of the present invention, Using the IBIFLAG system manufactured by Kodak Co., USA (see Kodak Co., Ltd. for details, see IBIFLAGE pitope. 1, 1, 1992). Polypeptide having 8 amino acids at the C-terminus of the peptide, that is, Asp Tyr Lys Asp Asp Asp Asp Lys, which is the amino acid sequence shown in SEQ ID NO: 7 in the sequence listing. An expression vector containing the gene encoding the polypeptide, which was subjected to the above method, was prepared in the same manner as described above, and a transformed cell line was obtained in the same manner. Fusion of the extracellular portion of the receptor tyrosine kinase of the present invention with FLAG from this cell culture supernatant by affinity chromatography using Anti-FLAGM 2 Affinity Ge1 manufactured by Kodak, USA The protein was purified.

The fusion protein of FLAG and the extracellular portion of the receptor tyrosine kinase of the present invention purified as described above was applied to a Ba1b / c mouse (manufactured by Japan SLC, Japan). Was immunized subcutaneously and intradermally. After two immunizations, blood was collected from the fundus and an increase in the antibody titer in the serum was observed. After the third immunization, the spleen cells of the mouse were removed, and the mouse myeloma cell line P3X63 Cell fusion was performed using Ag 8 (ATCCTIB 9) and the polyethylene glycol method. A hybridoma was selected in a HAT medium (Japan Institute for Immunity and Biology, Japan), and an antibody recognizing the extracellular portion of the receptor tyrosine kinase of the present invention was produced in the medium by an enzyme-linked immunosorbent assay. High Puri Doma Three strains were separated. These hybridomas were named 38-1E, 66-3A and 68-3A. These three hybrid strains, 38 1E, 66 3A, and 68 3A, were deposited at the National Institute of Advanced Industrial Science and Technology, Institute of Biotechnology, Japan, under the deposit number FE RM. BP-4884, FE RM BP-4885, FE RM BP-48886 were deposited on January 11, 1994, respectively.

 Monoclonal antibodies were purified from these cell culture supernatants using an antibody purification kit MabrapG, manufactured by Pharmacia, Sweden, according to the attached instruction manual. Using the three types of monoclonal antibodies thus prepared, a gene-introduced cell line expressing the full-length receptor tyrosine kinase of the present invention and a cell disrupted UT-7 cell line were obtained. When the blot was performed, it was confirmed that the band of 130 kilodalton was specifically recognized, and a monoclonal antibody recognizing the receptor type 1 tyrosine kinase of the present invention was confirmed. Was established.

Example 9 Isolation of cells using an antibody that recognizes the receptor tyrosine kinase of the present invention

In order to show that the anti-receptor tyrosine kinase antibody of the present invention is an antibody capable of detecting hematopoietic undifferentiated cells, the similarity with the anti-CD34 antibody detecting hematopoietic undifferentiated cells was examined. investigated. To 8 O ml of cord blood, 3 ml of a silica suspension (Immune Biological Research Institute, Japan) was added, and the mixture was incubated at 37 ° C for 1 hour. So After that, add 15 ml of Hank's saline (Gibco, USA) and add carohydrate. A mononuclear cell layer formed by centrifugation at 800 rpm for 20 minutes was collected, washed twice with Hanks 'saline, and washed with Hanks' saline twice (Pharmacia, Sweden). Cells were obtained. A suspension containing ⁵ × 105 cells of the cells was reacted with 100 μg / 1 of an anti-new receptor tyrosine kinase antibody on ice, and phycoerythrin-labeled anti-IgG After staining and washing with an antibody (Vecton Dickinson, USA), anti-CD34 antibody labeled with FITC, which was incubated in the presence of an IgG antibody (Vecton Dickinson, USA) (Vecton Dectonson, USA). These cells were measured with a Coulter flow cytometer EPICSELITE, UK. The measurement method of the flow cytometer followed the attached manual. As a result, the positive cells against the 38-1E antibody, 66-13A antibody, 68-3A antibody and anti-CD34 antibody were 2.8%, 0.5% and 3.9, respectively. % And 4.0%. In addition, the ratio of CD34-positive cells in the anti-receptor tyrosine kinase antibody-positive cells of the present invention was 5 2 for each of the 38-1E antibody, 66-3A antibody and 68-3A antibody. %, 67%, and 56%, indicating that many cells restained with the anti-receptor tyrosine kinase antibody of the present invention detected undifferentiated blood cells. .

Further, the anti-receptor tyrosine kinase of the present invention It was confirmed that undifferentiated cells could be isolated from the body. To 35 ml of umbilical cord blood was added 3.5 ml of a silica suspension (Immunological Institute, Japan), and the mixture was incubated at 37 ° C for 1 hour. Then, apply 10 ml of Hanks saline (Gibco, USA) [], and layer the cell suspension on Ficoll Pak solution (Pharmacia, Sweden). The mononuclear cell layer formed by centrifugation at a rotation speed of 20 minutes was collected and washed twice with Hanks' physiological saline to obtain mononuclear cells. The cells of some 4 X 1 0 ⁴ or anti Li Scepter type tyrosine Shinkina over peptidase antibodies of the present invention the cell suspension containing 3 8 - 1 E or 6 8 - 3 A 1 0 0 μ g / m 1 After washing on ice and washing, the cells were washed with a microselector (anti-mouse IgG antibody coating) from Applied Immuno Sciences (AIS), USA. Cells reacted with the anti-receptor type 1 tyrosine kinase antibody of the present invention were separated by the Panjung method. The micro selector was purchased from Asahi Medical Co., Ltd., Japan, and its use was performed according to the attached manual. As a control, mononuclear cells were separated in the same manner using an anti-CD34 antibody (Anti-HPCA-1 antibody, manufactured by Vecton Deckinson, USA). Using the obtained cells, the colony forming ability of the semi-solid medium was measured. The semi-solid medium used was a medium from Terrifox, Canada, and its use was in accordance with the manual issued by Terrifox. That is, the cells are suspended in a semi-solid medium and then placed in four 35 milliliter dishes (US, 1% each, and cultured for 14 days under conditions of 5% carbon dioxide, 95% air, 37 ° C, and high humidity. The number of colonies formed was measured under a microscope. According to the above procedure, the colonies of the 38-1E antibody, the 68-3 antibody and the anti-0034 antibody were 157, 182 and 113, respectively. That is, 38-1E and 68-3, which are the anti-receptor tyrosine kinase antibodies of the present invention, are anti-tyrosine kinase antibodies. Blood undifferentiated cells having much higher colony forming ability were isolated than blood undifferentiated cells isolated with the 034 antibody.

Example 10 Screening of megakaryocyte differentiation factor using the receptor type 1 thymic synkinase of the present invention Human fetal fibroblast cell line MR C-5 (RIKEN, Japan, Cell Development Bank, Japan) CDNA library prepared from mRNA of No. RCB0211) from a culture supernatant of cells into which a portion of DNA has been introduced in megakaryocyte leukemia cell line UT-7 The screening of a substance that reduces the expression of the receptor tyrosine kinase of the present invention was performed.

The expression library was prepared using Toyobo's Osaka Yamaichi Bergc DNA Library synthesis Kit. The presence or absence of the receptor tyrosine kinase of the present invention on UT-7 was determined by a flow cytometer using the monoclonal antibody established in Example 8 (EPICSE lite, United States, USA). Going at one Luther Was.

UT and cultured under the conditions described in Example 1 - 7 were adjust as the final cell number force ^{S 2 X 1 0 5 eells /} ml, COS expression library over produced by the above-described method against thereto Add the culture supernatant of the transformed cells created by gene transfer into the cells, collect the cells after culturing for 2 days, and reduce the expression of the receptor tyrosine kinase of the present invention on UT-7. The library was closed. As a result, COS cell clones that produce culture supernatants with reduced expression of the receptor type 1 thymic synthase of the present invention on UT-7 were obtained. When the insert was recovered and the insert gene sequence was examined, it was identical to the already known gene, human interleukin 1a (IL-11α). According to past studies, IL-11 is known to be involved in the colony formation of megakaryocyte cells (Briddelleta 1., B1ood 79: 332, 1992; Takahashieta 1., Br. J Haematol. 78: 480, 1991). These results indicate that by using the receptor tyrosine kinase of the present invention, it is possible to search for a bioactive substance that acts on megakaryocyte cells and is involved in increasing platelets. Was done.

 Industrial applicability

The receptor tyrosine kinase of the present invention is expressed in undifferentiated blood cells, but its expression level decreases with the differentiation of undifferentiated blood cells. Therefore, the receptor tyrosine of the present invention By using an antibody reactive with a kinase, undifferentiated blood cells, which are contained in a biological sample and have the receptor tyrosine kinase bound to the cell surface, are isolated from the sample. Thus, undifferentiated blood cells as described above contained in a biological sample can be specifically detected. The above method is useful not only for elucidating the mechanism of differentiation and proliferation of undifferentiated blood cells, but also for isolated hematopoietic stem cells expressing the receptor tyrosine kinase of the present invention. Undifferentiated blood cells can be applied to bone marrow transplantation, peripheral blood stem cell transplantation, and umbilical cord blood stem cell transplantation that are currently being performed. Furthermore, undifferentiated blood cell differentiation using the receptor tyrosine kinase of the present invention can inhibit or activate at least its receptor tyrosine kinase activity or suppress its expression. Screening of chemicals such as growth factors is also possible.

Sequence Listing SEQ ID NO: 1

Array length: 5 2 2

Sequence type: amino acid

Topology: linear

Sequence type: Protein

Origin

 Organism name: human

Array

 Leu Glu Glu u Thr Leu Leu Asn Tbr Lys Leu Glu Thr Ala Asp Leu Lys

1 5 10 15

Trp Va 1 Thr Phe Pro Gin Val Asp Gly Gin Trp Glu Glu Leu Ser Gly

 20 25 30

 Leu Asp Glu Glu Gin His Ser Val Arg Thr Tyr Glu Val Cys Asp Va 1

 35 40 45

 Gin Arg Ala Pro Gly Gin Ala His Trp Leu Arg Thr Gly Trp Val Pro

50 55 60

 Arg Arg Gly Ala Val His Val Tyr Ala Thr Leu Arg Phe Thr Met Leu 65 70 75 80

Glu Cys Leu Ser Leu Pro Arg Ala Gly Arg Ser Cys Lys Glu Thr Phe

 85 90 95

Thr Val Phe Tyr Tyr Glu Ser Asp Ala Asp Thr Ala Thr Ala Leu Thr

 100 105 110

 Pro Ala Trp Met Glu Asn Pro Tyr lie Lys Val Asp Thr Val Ala Ala

 115 120 125

 Glu His Leu Tht Arg Lys Arg Pro Gly Ala Glu Ala Thr Gly Lys Val

130 135 140

Asn Val Lys Tbr Leu Arg Leu Gly Pro Leu Ser Lys Ala Gi y Phe Tyr 145 150 155 160 Leu A 1 a Phe Gin Asp Gin Gly Ala Cys Met Ala Leu Leu Ser r Leu His

165 170 175

Leu Phe Tyr Lys Lys Cys Ala Gin Leu Thr Val Asn Leu Thr Arg Phe

 180 185 190

 Pro Glu Thr Val Pro Arg Glu Leu Val Val Pro Val Ala Gly Ser Cys

 195 200 205

 Val Val Asp Ala Val Pro Ala Pro Gly Pro Ser Pro Ser Leu Tyr Cys

210 215 220

 Arg Glu Asp Gly Gin Trp Ala Glu Gin Pro Val Thr Gly Cys Ser Cys 225 230 235 240

Ala Pro Gly Phe Glu Ala Ala Glu Gly Asn Thr Lys Cys Arg Ala Cys

 245 250 255

Ala Gin Gly Thr Phe Lys Pro Leu Ser Gly Glu Gly Set Cys Gin Pro

 260 265 270

 Cys Pro Ala Asn Ser His Ser Asn Thrlie Gly Ser Ala Val Cys Gin

 275 280 285

 Cys Arg Val Gly Tyr Phe Arg Ala Arg Thr Asp Pro Arg Gly Ala Pro

290 295 300

 Cys Thr Thr Pro Pro Ser Ala Pro Arg Ser Val Val Ser Arg Leu Asn 305 310 315 320

Gly Ser Sei Leu His Leu Glu Trp Ser Ala Pro Leu Glu Ser Gly Gl

 325 330 335

Arg Glu Asp Leu Thr Tyr Ala Leu Aig Cys Arg Glu Cys Arg Pio Gly

 340 345 350

 Gly Ser Cys Ala Pro Cys Gly Gly Asp Leu Thr Phe Asp Pro Gly Pro

 355 360 365

 Arg Asp Leu Val Glu Pro Trp Val Val Val Arg Gly Leu Arg Pro Asp

370 375 380

Phe Thr Tyr Thr Phe Glu Val Thr Ala Leu Asn Gly Val Ser Ser Leu 385 390 395 400

Ala Thr Gly Pro Va 1 Pro Phe Glu Pro Val Asn Val Thr Thr Asp Arg

 405 410 415

Glu Val Pro Pro Ala Val Ser Asp lie Arg Val Thr Arg Ser Ser Pro

 420 425 430

 Ser Ser Leu Ser Leu Ala Trp Ala Va 1 Pro Arg Ala Pro Ser Gly Ala

 435 440 445

 Val Leu Asp Tyr Glu Val Lys Tyr His Glu Lys Gly Ala Glu Gly Pro

450 455 460

 Ser Ser Val Arg Phe Leu Lys Thr Ser Glu Asn Arg Ala Glu Leu Arg 465 470 475 480

Gl Leu Lys Arg Gly Ala Ser T r Leu Val Gin Val Arg Ala Aig Ser

 485 490 495

Glu Ala Gly Tyr Gly Pio Phe Gl Gin Glu His His Sei Gin Thr Gin

 500 505 510

 Leu Asp Glu Ser Glu Gly Trp Arg Glu Gin

 515 520 SEQ ID NO: 2

Array length: 2 6 0

Sequence type: amino acid

 Topology: linear

Sequence type: Protein

Origin

 Organism name: human

Array

 Val Lys lie Glu Glu Val lie Gly Ala Gly Glu Phe Gl Glu Val Cys

1 5 10 15

Arg Gl Arg Leu Lys Ala Pro Gly Lys Lys Glu Ser Cys Val Ala lie > ―

DC

-

Va 1 S e r A 1 a Leu

 260 SEQ ID NO: 3

Array length: 9 7 2

Sequence type: amino acid

Number of chains: single strand

 Topology: linear

Sequence type: amino acid

Origin

 Organism name: human

Array

 Leu G 1 u G 1 u Th r Leu Leu As n Th r Lys Leu G 1 u Th r Ala Asp Leu Ly s

1 5 10 15

Trp Va 1 Thr Phe Pro Gin Va 1 Asp Gly Gin Trp Glu Glu Leu Ser Gl

 20 25 30

 Leu Asp Glu Glu Gin His Ser Va I Arg Thr Tyr Glu Va 1 Cys Asp Va 1

 35 40 45

 Gin Arg Ala Pro Gly Gin Ala His Trp Leu Arg Thr Gl Trp Va 1 Pro

50 55 60

 Arg Arg Gl Ala Va 1 His Va 1 Tyr Ala Thr Leu Arg Phe Thr Met Leu 65 70 75 80

Glu Cys Leu Ser Leu Pro Arg Ala Gl Arg Ser Cys Lys Glu Thr Phe

 85 90 95

Thr Va 1 Phe Tyr Tyr Glu Ser Asp Ala Asp Thr Ala Thr Ala Leu Thr

 100 105 110

 Pro Ala Trp Met Glu Asn Pro Tyr lie Lys Va 1 Asp Thr Va 1 Ala Ala

 115 120 125

Glu His Leu Thr Arg Lys Arg Pro Gly Ala Glu Ala Thr Gl Lys Va 1 130 135 140

Asn Va 1 Lys Thr Leu Arg Leu Gly Pro Leu Ser r Lys Ala Gly Phe Tyr 145 150 155 160

Leu Ala Phe Gin Asp Gin Gl Ala Cys Met Ala Leu Leu Ser Leu His

 165 170 175

Leu Phe Tyr Lys Lys Cys Ala Gin Leu Thr Va 1 Asn Leu Thr Arg Phe

 180 185 190

 Pro Glu Thr Va I Pro Arg Glu Leu Val Val Pro Val Ala Gly Ser Cys

 195 200 205

 Va 1 Val Asp Ala Val Pro Ala Pro Gly Pro Ser Pro Sei Leu Tyr Cys

210 215 220

 Arg Glu Asp Gly Gin Tip Ala Glu Gin Pro Va 1 Thr Gly Cys Ser Cys 225 230 235 240

Ala Pro Gly Phe Glu Ala Ala Glu Gly Aso Thr Lys Cys Arg Ala Cys

 245 250 255

Ala Gin Gly Thr Phe Lys Pro Leu Ser Gly Glu Gly Gly Ser Cys Gin Pro

 260 265 270

 Cys Pro Ala Asn Ser His Ser Asn Thrlie Gly Ser Ala Val Cys Gin

 275 280 285

 Cys Arg Val Gly Tyr Phe Arg Ala Arg Thr Asp Pro Arg Gly Ala Pro

290 295 300

 Cys Thr Thr Pro Pro Ser Ala Pro Arg Ser Val Val Ser Arg Leu Asn 305 310 315 320

Gl Ser Ser Leu His Leu Glu Trp Ser Ala Pro Leu Glu Ser Gly Gly

 325 330 335

Arg Glu Asp Leu Thr Tyr Ala Leu Arg Cys Arg Glu Cys Arg Pro Gly

 340 345, 350

 Gly Ser Cys Ala Pro Cys Gly Gly Asp Leu Thr Phe Asp Pro Gly Pro

355 360 365 B IV O 31 I 1 ΒΛ "10 ⁿ I3 I Λ J3S I dsy 3 | I njg

069 S8S 089

e IV Hi ni 3 JV I ΒΛ ^e lV njo asy o】 d dsy njg 丄 iqi ajo J j dsy

S! S OiS S9S

 311 "1. DO Λ s ^ l JRl 3 S! H lD 31 I n ^ l" 1 ° I0 ^! O «! Η dsy

099 S99 099

"s" 丄 B iv nig V 13 nsy "s ajg sq V ⁿ³ ls ^ O naq Λ

 O ^ S S £ S 0S9

MV Ι ΒΛ I ΒΛ a 11 Λ I ΒΛ ΠΛ neq ι ΒΛ 1 ^Β Λ ^ 10 IU ΡΛ e IV l

 SZ9 OZS SIS

10 E IV 31 I "31 V ⁿ³ 1 110 ⁿ I9 3iV d JI ig n ig] a $ dsy naq

019 90S 009

 aiO 丄 aiO u S; H S! H "19 aiO IO aqj OJ J jg ιχl9 B iV" 10

S6f 06 ^ 58 ^

J 3S 3 JV E I V 3iV I ΕΛ iIO I U naq 丄: ss MV ^ ID s ^ l na X i

0 9Zf 0 99 ^

2iy naq njg E iv 3】 V ^0S V "10 丄" 丄^η3 Ί ³ m V Λ ¹⁹ S ^ja S

 09 SS ^ OS ^ oi J I9 "IO MV JtIO" 19 s? H s q [ΒΛ "ID dsy naq A

 9 ^ on SS ^

B IV ^ 10 ^J3 S o ^ d eiV 3 JV ° HAEIV i ^ IV n ^ l ias ^η3 Ί i3S

 οε ^ s m

oij J8S SV iqi I ΕΛ V "I d ^S V \ E JV OJJ oi j A" 10

SO SO ^ v dsy qi iqi Ι ^ Λ ^ns V Λ ^{01 in n} I3 ⁰¹ i \ U ⁰ⁱ i ^ IO J l V

OOf 968 06S 988 naq las J 3 SA ^ 10 ^AS V neq B JV iqi Λ niO sqj 丄 laj

0Π S2 £ OZC dsy oJd 3 J v;) 3】 V eight Λ I ^Β U d U njg eight na dsy 3 JV

 Z 9

S £ 0r0 / f6df / XDd 98 £ SI / S6 OAV SSS efc / vIDd 98 € US6AVS O

CO

S e r Leu His G 1 n Leu Met Leu As p Cys T r p Gin Lys Asp A r g As n Ala

835 840 845

 Ar g Pro Ar g Phe Pro Gin Va 1 Va 1 Ser Ala Leu Asp Lys Met lie Ar g

850 855 860

 Asn Pro Ala Ser Leu Lys lie Va 1 Ala Arg Glu Asn Gly Gly Ala Ser 865 870 875 880

His Pro Leu Leu Asp Gin Arg Gin Pro His Tyr Ser Ala Phe Gly Ser

 885 890 895

Va 1 Gly Glu Trp Leu Arg Ala lie Lys Met Gly Arg Tyr Glu Glu Ser

 900 905 910

 Phe Ala Ala Ala Gly Phe Gly Ser Phe Glu Leu Va 1 Ser Gin lie Ser

 915 920 925

 Ala Glu Asp Leu Leu Arg lie Gl Va 1 Thi Leu Ala Gl His Gin Lys

930 935 940

 Lys lie Leu Ala Ser Va 1 Gin His Met Lys Ser Gin Ala Lys Pro Gl 945 950 955 960

Thr Pro Gly Gly Thr Gly Gly Pro Ala Pro Gin Tyr

 965 970 SEQ ID NO: 4

Sequence length: 4 2 9 0 and 9 8 7

Sequence type: nucleic acid and amino acid

Number of chains: double-stranded and single-stranded

Topology: linear

Sequence type: cDNA to mRNA, and amino acid

Origin

 Organism name: human

However, the gene sequence is undecided for the gene sequence X.

Array GGGCTGGCG GGGAGGGAAC ACAGGTCAGT GTGGGGACAG GGGTCACGGT 49 GGACACGGGG GTGGGCTGTC TCAGGGGGGG AGACCGCGAG CGGCCGGCTC AGCCCCCGCC 109 ACCCGGGGCG GGACCCCGAG GCCCCGGAGG GACCCCAACT CCAGCCACGT CTTGCTGCGC 169 GCCCGCCCGG CGCGGCCACT GCCAGCACGC TCCGGGCCCG CCGCCCGCGC GCGCGCACAG 229 ACGCGGGGCC ACACTTGGCG CCGCCGCCCG GTGCCCCGCA CGCTCGCATG GGCCCGCGCT 289 GAGGGCCCGA CGAGGAGTCC CGCGCGGAGT ATCGGCGTCC ACCCGCCCAG GGAGAGTCAG 349 ACCTXXXXXG GCGAGGGCCC CCCAAACTCA GTTCGGATCC TACCCGAGTG AGGCGGCGCC 409 ATG GAG CTC CGG GTG CTG CTC TGC TGG GCT TCG TTG GCC GCA GCT TTG 457 Met Glu Leu Arg Val Leu Leu Cys Tip Ala Ser Leu A 1 a Ala A 1 a Leu -15 -10 -5-1 1

GAA GAG ACC CTG CTG AAC ACA AAA TTG GAA ACT GCT GAT CTG AAG TGG 505 Glu Glu Thr Leu Leu Asn Thr Lys Leu Glu Thr Ala Asp Leu Lys Trp

 5 10 15 GTG ACA TTC CCT CAG GTG GAC GGG CAG TGG GAG GAA CTG AGC GGC CTG 553 Val Thr Pbe Pro Gin Val Asp Gly Gin Trp Glu Glu Leu Ser Gl Leu

 20 25 30

GAT GAG GAA CAG CAC AGC GTG CGC ACC TAC GAA GTG TGT GAC GTG CAG 601 Asp Glu Glu Gin His Ser Val Arg Thr Tyr Glu Val Cys Asp Val Gin

 35 40 45

CGT GCC CCG GGC CAG GCC CAC TGG CTT CGC ACA GGT TGG GTC CCA CGG 649 Arg Ala Pro Gl Gin Ala His Trp Leu Arg Thr Gly Trp Val Pro Arg 50 55 60 65

CGG GGC GCC GTC CAC GTG TAC GCC ACG CTG CGC TTC ACC ATG CTC GAG 697 Arg Gly Ala Val His Val Tyr Ala Thr Leu Arg Phe Thr Me t Leu Glu

 70 75 80

TGC CTG TCC CTG CCT CGG GCT GGG CGC TCC TGC AAG GAG ACC TTC ACC 745 Cys Leu Ser Leu Pro Arg Ala Gl Arg Ser Cys Lys Glu Thr Phe Thr

85 90 95 GTC TTC TAC TAT GAG AGC GAT GCG GAC ACG GCC ACG GCC CTC ACG CCA 793 SZ 092

B IV e I v 8 JV ssl iqi asy "3 niO" 10 s¾d O ⁰

9ΠΙ 303 101 0D3 VOO 001 3VV 3DV OVV 900 OVD 1D9 VDO OVO :) 丄 丄 3!);) 033

 B I V sX3 Jag iqi ° I0 nig E IV "丄 ai9" 3 dsV n i

LLU 丄;) 3 丄 ί) 丄 33V D01 000 3DV;) 丄 3 ODD OVD VVO DDO 001 3V3 000 丄 V3 OVO m QZZ

3iV "丄 n 97 lag oid ° ^J d E IV" JJ dsv Λ

6Πΐ 100 DDI DV1 DID 00V 3D0 09V ODD 003 13D 000 DDD D19 030 IVO f) 丄;)

 S02 OOZ 961

I ΒΛ s ^ O J 8S C Ι «Λ I ΕΛ ° \ n

 1801 913 33 丄 3V 100 ODD;) 丄;) 0D3 310 tL 0 0 OVO 003 丄 V OVO

 061 981 081

 OJ <J aqj 3JV 丄 asv ΒΛ ΒΛ JU rial ai9 E IV sXo J "aid

C £ OI ODD Oil VOO 13V;) 丄;) OVV 013 IOV 91D OVD 000 301 OVV VVV DV1 Oil

 SZI Oil S9l

 naq s nai naq B IV s B IV dsy njg J V

986 310 OVD 9J.D DDI VJ-D 01D 300 01V 091 030 100 OVO OVO OVD Oil ODD

 091 SSI OSl

 Π3Ί "丄 aqj siq I as neq na jqi s q ι ΕΛ

I £ 6 010;) V 丄 3 丄 丄 000 1DD OVV GOV 010 030 VOO 310 103 010 00V OVV 010 SH OH 0Π n sy Λ s q 13 JRI V "ID V OIJ JU na s JH

688 丄 VV 010 3VV 030 33V 300 0V9 003 939 丄 3;) 090 3VV 033 DOV 010 丄 V3

 S21 021 SI l

"IS BIV B iv d sv I ΒΛ s I Jo I j asy" 19 i E iv 8 0V9 000 330 310 03V 3V0 010 OVV 31V 3V1 333 3VV OVO 01V 901 303 on 901 001

ε IV 丄 BIV ¹ m dsy ε IV d sy i8S ni J ”) aq <j I n

 9 9

ξ £ 0Ζ0Ι 6ά £ / Σ3ά 98CSI / S6 OW ―>-

>--3

405 410 415

GTA CCT CCT GCA GTG TCT GAC ATC CGG GTG ACG CGG TCC TCA CCC AGC 1753 Val Pro Pro Ala Val Ser Asp I 1 e Arg Va 1 Thr Arg Ser Ser Pro Ser

 420 425 430

AGC TTG AGC CTG GCC TGG GCT GTT CCC CGG GCA CCC AGT GGG GCT GTG 1801 Ser Leu Ser Leu Al a Trp Ala Val Pro Arg Ala Pro Ser Gly Ala Val

 435 440 445

 CTG GAC TAC GAG GTC AAA TAC CAT GAG AAG GGC GCC GAG GGT CCC AGC 1849 Leu Asp Tyi Glu Val Lys Tyr His Glu Lys Gly Ala Glu Gly Pio Ser 450 455 460 465

AGC GTG CGG TTC CTG AAG ACG TCk GAA AAC CGG GCA GAG CTG CGG GGG 1897 Ser Val Arg Phe Leu Lys Thr Ser Glu Asn Arg Ala Glu Leu Arg Gly

 470 475 480

CTG AAG CGG GGA GCC AGC TAC CTG GTG CAG GTA CGG GCG CGC TCT GAG 1945 Leu Lys Arg Gly Ala Ser Tyi Leu Val Gin Val Arg Ala Arg Ser Glu

 485 490 495

GCC GGC TAC GGG CCC TTC GGC CAG GAA CAT CAC AGC CAG ACC CAA CTG 1993 Ala Gly Tyr Gly Pro Phe Gly Gin Glu His His Ser Gin Thr Gin Leu

 500 505 510

GAT GAG AGC GAG GGC TGG CGG GAG CAG CTG GCC CTG ATT GCG GGC ACG 2041 Asp Glu Ser Glu Gl Trp Arg Glu Gin Leu Ala Leu He Ala Gly Thr

 515 520 525

 GCA GTC GTG GGT GTG GTC CTG GTC CTG GTG GTC ATT GTG GTC GCA GTT 2089 Ala Val Val Gly Val Val Leu Val Leu Val Val lie Val Val Ala Val 530 535 540 540 545

CTC TGC CTC AGG AAG CAG AGC AAT GGG AGA GAA GCA GAA TAT TCG GAC 2137 Leu Cy s Leu Arg Lys Gin Ser Asn Gly Arg Glu A 1 a Glu Tyr Ser Asp

 550 555 560

AAA CAC GGA CAG TAT CTC ATC GGA CAT GGT ACT AAG GTC TAC ATC GAC 2185 Lys His Gly Gin Tyr Leu He Gly His Gly Thr Lys Val Tyr I 1 e Asp

565 570 575

 CCC TTC ACT TAT GAA GAC CCT AAT GAG GCT GTG AGG GAA TTT GCA AAA 2233

Pro Phe Thr Tyr Glu Asp Pro Asn Glu Ala Va 1 Arg Glu Phe Ala Lys

 580 585 590

 GAG ATC GAT GTC TCC TAC GTC AAG ATT GAA GAG GTG ATT GGT GCA GGT 2281

Glu lie Asp Val Ser Tyr Va 1 Lys lie Glu Glu Va 1 lie Gly Ala Gly

 595 600 605

 GAG TTT GGC GAG GTG TGT CGG GGG CGG CTC AAG GCC CCA GGG AAG AAG 2329

Glu Phe Gly Glu Val Cys Arg Gly Arg Leu Lys Ala Pro Gly Lys Lys

610 615 620 625

GAG AGC TGT GTG GCA ATC AAG ACC CTG AAG GGT GGC TAC ACG GAG CGG 2377

Glu Ser Cys Va 1 Ala He Lys Thr Leu Lys Gly Gly Tyr Thr Glu Arg

 630 635 640

 CAG CGG CGT GAG TTT CTG AGC GAG GCC TCC ATC ATG GGC CAG TTC GAG 2425

Gin Arg Arg Glu Phe Leu Ser Glu Ala Ser lie Met Gly Gin Phe Glu

 645 650 655

 CAC CCC AAT ATC ATC CGC CTG GAG GGC GTG GTC ACC AAC AGC ATG CCC 2473

His Pro Asn He lie Arg Leu Glu Gly Val Val Thr Asn Ser Met Pio

 660 665 670

 GTC ATG ATT CTC ACA GAG TTC ATG GAG AAC GGC GCC CTG GAC TCC TTC 2521

Val Met 11 e Leu Thr Glu Phe Met Glu Asn Gly Ala Leu Asp Ser Phe

 675 680 685

 CTG CGG CTA AAC GAC GGA CAG TTC ACA GTC ATC CAG CTC GTG GGC ATG 2569

Leu Arg Leu Asn Asp Gly Gin Phe Thr Val lie Gin Leu Val Gly Met

690 695 700 705

CTG CGG GGC ATC GCC TCG GGC ATG CGG TAC CTT GCC GAG ATG AGC TAC 2617

Leu Arg Gly lie Ala Set Gly Met Arg Tyr Leu Ala Glu Met Ser Tyr

710 715 720 "S e iv 19 O asy n jo 3 jy E iv I eai I s naq ias BIV o ζ6οε 3VD VD1 DDO 000 300 1VV OVO 000 ODD 3 丄 3 DIV VVV;) 丄;) 30V 330 DDO 998 098 SS8 098 π sy 3JV 81 I dsy ne] y】 as Ι ΒΛ m aiO oid 3Rd 3】 V ojj

6ί-οε 3VV 003 Vpp V9IV OVV DV9 913 0D9 00V 010 013 9V0 ODD :) 丄 丄 33;) ODD

 OH S £ 8

3 JV B ivusv 3 JV dsy dsv naq i aj¾ naq DID s! H " ³ 1 ιοοε 090 while 3 1VV SOD 3V0 VVV 0V3 00 ox 3V9 013 31V 013 OVD 3V3 DID

 0E8 SZ8 0Z8

 J3S s dsy o O J J "丄 dsv αΐθ" 13 311

SS6Z 00V 030 101 OVO ODD ODD 013 003 0V1 OVO OVO VV3 丄 IV

 SI8 018 908

 Vas v 3 I I ΠΙ9 as dsy dix old 3JV "19 ^ 19

906 Ζ 330 丄 VV 3 丄 V 3 丄 3 DVO OVD 1VV 00V D1V OVO 991 DV1 ODD 99V 0V9 000

 008 961 061

 Λ «10 丄 MM IEA a ϊ I O dil eiv ds" S

ZS8Z VOX 01V 010 OVO; m iuv 910 1 丄 V 090 0V1 10V 931 330 丄 V3 丄 3V

S8Z 08Z SZZ Oil MV B IV 31 I Biv "ID oii MV iq 丄 dJ 丄 3iv

608 Ζ 0D3 DDI 1DV 011 9VV 093 Oil 033 UV DDO 9V0 030 DD3 丄 OV 001 V90

 S92 09i 991 I. 】 D I 10 neq ias "S 丄 j i" id dsy】 3s ΐ 3ξ

1922 D1V DDO I1V OVV V03 ¥ 30 3 丄 :) 301 DOV DOV 3V1 03V 333 1V0 331 101

 09Z

 n [0 Mi) 3iy i as i \ aqj dsy "s s l s o I ΕΛ

ΖΜΙ 3VV OVO 9VD 0 0 «L iL V03 331 11D 30D 111 DVO 1D1 910 VVV 301 310

 9Ci oez

 n 3 ΐ a s v J a s a s v I q n a q a [[u s y 2 ι V B I V ε I v naq dsy 3] v s! H Ι

S99Z :) 丄 :) DVV 3DV OVV 310 V 丄 :) :) 丄 V 3VV 003 100 130 3 丄 :) DVO V03 OVD 010

 0 9

<i £ 0Z0IP6d £ l Dd 870 875 880

 CCT CTC CTG GAC CAG CGG CAG CCT CAC TAC TCA GCT TTT GGC TCT GTG 3145

Pro Leu Leu Asp Gin Arg Gin Pro His Tyr Ser Ala Phe Gly Ser Val

 885 890 895

 GGC GAG TGG CTT CGG GCC ATC AAA ATG GGA AGA TAC GAA GAA AGT TTC 3193

Gly Glu Tip Leu Arg Ala lie Lys Met Gly Arg Tyr Glu Glu Ser Phe

 900 905 910

 GCA GCC GCT GGC TTT GGC TCC TTC GAG CTG GTC AGC CAG ATC TCT GCT 3241 Ala Ala Ala Gly Phe Gly Ser Phe Glu Leu Val Ser Ginlie Ser Ala

 915 920 925

 GAG GAC CTG CTC CGA ATC GGA GTC ACT CTG GCG GGA CAC CAG AAG AAA 3289

Glu Asp Leu Leu Ai g 11 e Gly Val Thr Leu Ala Gly His Gin Lys Lys 930 935 940 945

ATC TTG GCC AGT GTC CAG CAC ATG AAG TCC CAG GCC AAG CCG GGA ACC 3337 lie Leu Ala Ser Val Gin His Met Lys Ser Gin Ala Lys Pro Gly Thr

 950 955 960

CCG GGT GGG ACA GGA GGA CCG GCC CCG CAG TAC 3370 Pro Gly Gly Thr Gly Gly Pro Ala Pro Gin Tyr

 965 970

 TGACCTGCAG GAACTCCCCA CCCCAGGGAC ACCGCCTCCC CATTTTCCGG GGCAGAGTGG 3430

GGACTCACAG AGGCCCCCAG CCCTGTGCCC CGCTGGATTG CACTTTGAGC CCGTGGGGTG 3490

AGGAGTTGGC AATTTGGAGA GACAGGATTT GGGGGGTTCT GCCATAATAG GAGGGGAAAA 3550

TCACCCCCCC AGCCACCTCG GGGAACTCCA GACCAAGGGT GAGGGCGCCT TTCCCTCAGG 3610

ACTGGGTGTG ACCAGAGGAA AAGGAAGTGC CCAACATCTC CCAGCCTCCC CCAGGTGCCC 3670

CCCCTCACCT TGATGGGTGC GTTCCCGCAG ACCAAAGAGA GTGTGACTCC CTTGCCAGCT 3730

CCAGAGTGGG GGGGCTGTCC CAGGGGGCAA GAAGGGGTGT CAGGGCCCAG TGACAAAATC 3790

ATTGGGGTTT GTAGTCCCAA CTTGCTGCTG TCACCACCAA ACTCAATCAT TTTTTTCCCT 3850

TGTAAATGCC CCTCCCCCAG CTGCTGCCTT CATATTGAAG GTTTTTGAGT TTTGTTTTTG 3910

GTCTTAATTT TTCTCCCCGT TCCCTTTTTG TTTCTTCGTT TTGTTTTTCT ACCGTCCTTG 3970 TCATAACTTT GTGTTGGAGG GAACCTGTTT CACTATGGCC TCCTTTGCCC AAGTTGAAAC 4030

AGGGGCCCAT CATCATGTCT GTTTCCAGAA CAGTGCCTTG GTCATCCCAC ATCCCCGGAC 4090

CCCGCCTGGG ACCCCCAAGC TGTGTCCTAT GAAGGGGTGT GGGGTGAGGT AGTGAAAAGG 4150

GCGGTAGTTG GTGGTGGAAC CCAGAAACGG ACGCCGGTGC TTGGAGGGGT TCTTAAATTA 4210

TATTTAAAAA AGTAACTTTT TGTATAAATA AAAGAAAATG GGACGTGTCC CAGCTCCAGG 4270

GGTGAAAAAA AAAAAAAAAA 4290 SEQ ID NO: 5

Array length: 2 7

Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Sequence type: D N A

Origin: by chemical synthesis

Sequence: 5'-TTGTCGACAC (AC) G (AG) GA (CT) (CT) T (CG) GC (ACGT) GC (ACGT) (AC) G-3, SEQ ID NO: 6

Array length: 2 4

Sequence type: nucleic acid

Number of chains: single strand

 Topology: linear

Array typeD N A

Origin: by chemical synthesis

Sequence: 3'-CT (AG) CA (CG) ACC (AT) (CG) (AG) A (AT) ACCTTAAGGT-5 '

SEQ ID NO: 7

Sequence length: 2 7 and 8

Sequence type: nucleic acid and amino acid

Number of chains: single strand Topology: linear

Sequence type: DNA and amino acid

Origin: by chemical synthesis

Sequence: 5, -GAT TAT AAA GAT GAT GAT GAT AAA TGA-3 '

 Asp Tyr Lys Asp Asp Asp Asp Lys

Claims

The scope of the claims

 1. An isolated polypeptide having an receptor tyrosine kinase activity and containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2 and 3 in the sequence listing, or the polypeptide A homologous mutant of a peptide having receptor tyrosine kinase activity.

2. A polypeptide having an receptor tyrosine kinase activity and containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3 in the sequence listing, or a polypeptide of the polypeptide, Receptor An isolated DNA encoding a homologous variant having tyrosine kinase type 1 activity.

 3. DNA that has a receptor tyrosine kinase activity and encodes a polypeptide containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2 and 3 in the sequence listing A replicable recombinant DNA comprising a DNA encoding a homologous mutant of the mosquito or the polypeptide having a receptor tyrosine kinase activity, which is expressed in a replicable expression vector. .

4. Coding a polypeptide having an receptor tyrosine kinase activity and containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2 and 3 in the sequence listing D A replicable recombination in which a DNA encoding NA or a homologous mutant of the polypeptide having a receptor tyrosine kinase activity is expressed in a replicable expression vector. A microorganism or eukaryotic cell transformed with DNA.

5. A polypeptide having an receptor tyrosine kinase activity and containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2 and 3 in the sequence listing, or a receptor of the polypeptide. An antibody reactive with homologous mutants having tyrosine kinase activity.

 6. The antibody according to claim 5, which is fixed to a solid support.

7. A method for isolating somatic cells containing a polypeptide having receptor tyrosine kinase activity,

 (1) a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 3 in the sequence listing, or a homologue of the polypeptide having a tyrosine kinase activity, having a receptor tyrosine kinase activity The antibody according to claim 5 or 6 is contacted with a biological sample containing somatic cells having a mutant as an antigen on the surface thereof under conditions where the antigen and the antibody form an antigen-antibody complex. Thereby obtaining an antigen-antibody reaction mixture; and

(2) isolating the somatic cells containing the polypeptide or the homologous mutant in the form of an antigen-antibody complex from the antigen-antibody reaction mixture,

A method comprising:

 8. The method according to claim 8, wherein the biological sample is an aqueous suspension containing blood undifferentiated cells or a body fluid containing blood undifferentiated cells.

9. After the above step (2), the antibody is separated from the antigen-antibody complex. The method according to claim 7 or 8, wherein a somatic cell containing the polypeptide or the homologous variant in a form not conjugated to an antibody is obtained by separation.

 10. A method for detecting a polypeptide having receptor tyrosine kinase activity,

 (1) a polypeptide having an receptor tyrosine kinase activity and containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2 and 3 in the sequence listing, or a polypeptide of the same; The antibody according to claim 5 or 6 is contacted with a biological sample containing a homologous mutant having a tyrosin kinase activity as an antigen under conditions where the antigen and the antibody form an antigen-antibody complex. Thereby obtaining an antigen-antibody reaction mixture; and

 (2) detecting the polypeptide or the homologous mutant in the form of an antigen-antibody complex,

A method that encompasses this.

 11. The method according to claim 10, wherein the biological sample is an aqueous suspension containing undifferentiated blood cells or a body fluid containing undifferentiated blood cells. Method.

 12. A method of screening for a substance capable of inhibiting or activating tyrosine kinase activity or a chemical substance that suppresses the expression of a polypeptide having tyrosine kinase activity.

(1) The sample material is used for receptor tyrosine kinase activity. Or an isolated polypeptide containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3, or a phase of the polypeptide having receptor tyrosine kinase activity The polypeptide is contacted with the mutant, and the polypeptide or the homologous mutant has an inhibitory or activating ability for the receptor tyrosine kinase activity of the polypeptide or the homologous mutant, or the expression suppressing ability of the polypeptide or the homologous mutant as an index. At least detecting a chemical substance that inhibits or activates the receptor tyrosine kinase activity of the polypeptide or the homologous variant or suppresses the expression of the polypeptide or the homologous variant; and

 (2) A method comprising isolating a detected chemical substance from a sample material.

 13. An isolation selected from the group consisting of a sense DNA containing at least 12 consecutive nucleotide sequences of the nucleotide sequence of SEQ ID NO: 4 in the sequence listing and an antisense DNA complementary to the sense DNA. Obtained by subjecting the obtained DNA fragment, and the sense DNA and the antisense DNA to methylation, methylphosphate formation, thiophosphate formation, or deamination, respectively. An isolated DNA fragment selected from the group consisting of a sense DNA and a derivative of the antisense DNA.

1 4. Isolation selected from the group consisting of a sense RNA containing at least 12 consecutive nucleotide sequences of the nucleotide sequence of SEQ ID NO: 4 in the sequence listing and an antisense RNA complementary to the sense RNA The obtained RNA fragment, and the antisense RNA and the sense RNA, respectively, by methylation, methylphosphate formation, thiophosphoformation, or deamination. An isolated RNA fragment selected from the group consisting of an antisense RNA and a derivative of the sense RNA.