WO1995015386A1 - Nouvelle tyrosine-kinase receptrice - Google Patents

Nouvelle tyrosine-kinase receptrice Download PDF

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Publication number
WO1995015386A1
WO1995015386A1 PCT/JP1994/002035 JP9402035W WO9515386A1 WO 1995015386 A1 WO1995015386 A1 WO 1995015386A1 JP 9402035 W JP9402035 W JP 9402035W WO 9515386 A1 WO9515386 A1 WO 9515386A1
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Prior art keywords
tyrosine kinase
polypeptide
receptor tyrosine
antibody
dna
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PCT/JP1994/002035
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English (en)
Japanese (ja)
Inventor
Seiji Sakano
Mitsuharu Oono
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Asahi Kasei Kogyo Kabushiki Kaisha
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Application filed by Asahi Kasei Kogyo Kabushiki Kaisha filed Critical Asahi Kasei Kogyo Kabushiki Kaisha
Priority to JP51552895A priority Critical patent/JP3665071B2/ja
Priority to AU11208/95A priority patent/AU1120895A/en
Publication of WO1995015386A1 publication Critical patent/WO1995015386A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes

Definitions

  • 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.
  • 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.
  • red blood cells carry oxygen in the body
  • platelets provide a haemostatic effect
  • 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.
  • EPO Erythropoietin
  • G-CSF granulocyte colony-stimulating factor
  • 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.
  • the tyrosine kinase gene may 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.
  • tyrosine kinases Very conserved sequences are among the amino acid sequences
  • New tyrosine kinase gene fragment can be obtained when used as a primer in the PCR method.
  • 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.
  • SCF stem cell factor
  • 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.
  • a human megakaryoblastic leukemia cell line UT_7 obtained from Lecturer, Department of Hematology, Jichi Medical University, Jichi Medical University, Japan
  • GM-CSF granulocyte macrophage-colony stimulating factor
  • IL-3 interleukin 3
  • UT-7 cells 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.
  • a novel receptor-type tyrosine kinase was found in which the expression of mR ⁇ was strongly observed in the undifferentiated state of U ⁇ -7 cells, but the expression was not observed with the differentiation into megakaryocytes. .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a homologous variant of the polypeptide having a receptor tyrosine kinase activity is provided.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the usual test book mentioned above is, for example,
  • 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.
  • 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.
  • 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.
  • the labeling method for isotope isotope
  • [ 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.
  • 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
  • the intracellular portion composed of 4 2 4 amino acid, which is the second component, is composed of the same.
  • 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.
  • 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 benhuieta, One ogene, 7, 2499-2506, 1992), chicken C ek — 5
  • 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.
  • a transformant can be obtained by incorporating a microorganism or a eukaryotic cell (eg, an insect cell or an animal cell) as a host.
  • 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.
  • 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.
  • a similar phenomenon can be confirmed in other megakaryoblast cell lines, since the expression of mRNA is not observed upon differentiation into megakaryocytes.
  • 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.
  • an antibody against the extracellular portion (SEQ ID NO: 1) is a very effective means that can detect cells in a living state.
  • 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.
  • 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.
  • Example 8 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.
  • 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.
  • 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.
  • 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.
  • a method for isolating a somatic cell containing a polypeptide having a receptor tyrosine kinase activity comprising:
  • 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
  • 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.
  • 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.
  • cells expressing the receptor tyrosine kinase of the present invention can be isolated by digesting the antigen or antibody with an enzyme.
  • 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.
  • a receptor-type tyro As a method for detecting a polypeptide having a synkinase activity,
  • 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
  • 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.
  • a method using a flow site meter is generally used, but other known detection methods utilizing an antigen-antibody reaction can also be used.
  • 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.
  • 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
  • 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,
  • 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;
  • a method which comprises isolating a detected chemical substance from a sample material.
  • the present invention binds to the receptor tyrosine kinase of the present invention and suppresses or promotes the autophosphorylation of the receptor tyrosine kinase.
  • 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-1 ⁇ ) which is a megakaryocyte differentiation factor as shown in FIG.
  • 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 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.
  • 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.
  • Example 1 Po1y of human megakaryoblastic leukemia cell line UT-7
  • 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
  • IMDM Iscove's modified Dulbecco's medium
  • FCS 10% fetal calf serum
  • FCS fetal calf serum
  • h GM- CSF human granulocyte macro off ⁇ Jiko b knee stimulating factor
  • RNA extraction 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.
  • PMA phorbol 12-millistate 13-acetate
  • Lithium chloride Z urea method LithiumCh1oride / Urea method: Eur.J.Biochem. 107: 303
  • 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.
  • 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.
  • 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 ⁇ g / ⁇ 1 and used for PCR.
  • 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 ⁇ l of deionized water, and 10X buffer solution (500 mM KC 1, 100 mM MTris-HC 1
  • 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 ⁇ l, dNTPMixture (Takara Shuzo, Japan) 6.4 ⁇ l, sense primer specific for tyrosine kinase described above ⁇ TKI (lg / jul, 1.5 ⁇ l) and 0.2 ⁇ l of Taq DNA polymerase (Amp1iTaq, manufactured by PerkinElmer Inc., USA: 5 U_iul) (described in SEQ ID No. 5 in the column list).
  • 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.
  • TE-saturated phenol Nippon Gene, Japan
  • the upper layer was separated, and the same separation operation was performed using a TE-saturated phenol: chloroform (1: 1) solution and then chloroform. .
  • 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.
  • pBluescript IIKS manufactured by Stratagene, USA; hereinafter, referred to as pB1uescript
  • 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 ⁇ g / ml ampicillin.
  • 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.
  • 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.
  • 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).
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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 was purified from the DNA labeling kit (MegaPrime kit).
  • DNA labelingsystem Amasi, UK At catcher arm Ltd.
  • MOLT-4 available from ATCC, CCL1582.
  • 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
  • An expression plasmid was constructed by connecting the y (A) + signal and the dihydrofolate reductase gene. This plasmid was purified, and 20 ⁇ g 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.
  • 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.
  • a band of about 80 kDa protein was confirmed in the cell culture supernatant.
  • a protein band of 30 kDa and 130 kDa was confirmed in the cell lysate, respectively.
  • 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.
  • 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
  • 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 ⁇ g / 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.
  • 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%.
  • 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. .
  • 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.
  • a silica suspension Immunological Institute, Japan
  • 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 ⁇ g / m 1
  • a microselector anti-mouse IgG antibody coating
  • AIS Applied Immuno Sciences
  • 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.
  • mononuclear cells were separated in the same manner using an anti-CD34 antibody (Anti-HPCA-1 antibody, manufactured by Vecton Deckinson, USA).
  • Anti-HPCA-1 antibody manufactured by Vecton Deckinson, USA.
  • 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.
  • 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.
  • Example 1 - 7 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.
  • IL-11 ⁇ human interleukin 1a
  • 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).
  • 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
  • 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.
  • 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.
  • Organism name human
  • Organism name human
  • Val Lys lie Glu Glu Val lie Gly Ala Gly Glu Phe Gl Glu Val Cys
  • Organism name human
  • Trp Va 1 Thr Phe Pro Gin Va 1 Asp Gly Gin Trp Glu Glu Leu Ser Gl
  • Lys lie Leu Ala Ser Va 1 Gin His Met Lys Ser Gin Ala Lys Pro Gl 945 950 955 960
  • Organism name human
  • Gin Arg Arg Glu Phe Leu Ser Glu Ala Ser lie Met Gly Gin Phe Glu
  • Sequence type nucleic acid
  • Sequence type nucleic acid

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Abstract

Nouvelle tyrosine-kinase réceptrice s'exprimant dans une cellule sanguine non différenciée mais subissant une réduction du taux d'expression à mesure que progresse la différenciation dans la cellule non différenciée; et anticorps réagissant avec ladite kinase. L'utilisation de cet anticorps permet d'isoler une cellule sanguine non différenciée renfermant ladite kinase liée à sa surface, à partir d'un échantillon biologique contenant une cellule de ce type, et de détecter de manière spécifique une telle cellule comprise dans un échantillon biologique. On a également prévu un procédé de détection d'une substance chimique susceptible au moins d'inhiber ou d'activer l'activité de la tyrosine-kinase réceptrice, ou de s'opposer à l'expression de la kinase. En outre, on a prévu un ADN codant pour ladite kinase; un ADN recombiné pouvant subir la réplication et étant obtenu par intégration expressible dudit ADN dans un vecteur d'expression pouvant subir la réplication; un micro-organisme ou une cellule eucaryote transformé(e) par ledit ADN recombiné; un fragment d'ADN sens, un fragment d'ADN anti-sens et leurs dérivés, chacun étant préparé par utilisation d'un ADNc codant pour ladite kinase; ainsi qu'un fragment d'ARN sens, un fragment d'ARN anti-sens et leurs dérivés.
PCT/JP1994/002035 1993-12-02 1994-12-02 Nouvelle tyrosine-kinase receptrice WO1995015386A1 (fr)

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JP51552895A JP3665071B2 (ja) 1993-12-02 1994-12-02 新規なリセプター型チロシンキナーゼ
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015174443A1 (fr) * 2014-05-14 2015-11-19 国立大学法人東京大学 Promoteur d'induction de différenciation mégacaryocytaire et de production de plaquettes contenant de l'interleukine 1α (il-1alpha)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993015201A1 (fr) * 1992-01-22 1993-08-05 New England Deaconess Hospital Nouvelles tyrosine kinases de proteines

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993015201A1 (fr) * 1992-01-22 1993-08-05 New England Deaconess Hospital Nouvelles tyrosine kinases de proteines

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
J. BIOL. CHEM., Vol. 269, No. 19, 1994, BENNETT BRIAN D. et al., "Cloning and Characterization of HTK, a Novel Transmembrane Tyrosine Kinase of the EPH Subfamily", pages 14211-18. *
NATURE, Vol. 324, No. 13, 1986, RANDALL K. SAIKI et al., "Analysis of Enzymatically Amplified beta-Globin and HLA-DQalpha DNA With Allele-specific Oligonuclectide Probes", pages 163-166. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015174443A1 (fr) * 2014-05-14 2015-11-19 国立大学法人東京大学 Promoteur d'induction de différenciation mégacaryocytaire et de production de plaquettes contenant de l'interleukine 1α (il-1alpha)

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