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WO1998026071A1 - Chemokine cc humaine elc - Google Patents

Chemokine cc humaine elc Download PDF

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Publication number
WO1998026071A1
WO1998026071A1 PCT/JP1997/004573 JP9704573W WO9826071A1 WO 1998026071 A1 WO1998026071 A1 WO 1998026071A1 JP 9704573 W JP9704573 W JP 9704573W WO 9826071 A1 WO9826071 A1 WO 9826071A1
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Prior art keywords
protein
human
sequence
elc
amino acid
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PCT/JP1997/004573
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English (en)
Japanese (ja)
Inventor
Ryu Yoshida
Toshio Imai
Osamu Yoshie
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Shionogi & Co., Ltd.
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Priority to AU54106/98A priority Critical patent/AU5410698A/en
Publication of WO1998026071A1 publication Critical patent/WO1998026071A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2

Definitions

  • the present invention relates to a novel human CC-type chemokine, a polynucleotide molecule encoding the protein, a method for producing the protein, a pharmaceutical composition containing the protein, a polynucleotide encoding the protein, and the like.
  • the present invention relates to a method for screening agonist Z inverse agonist Z antagonist for biological action induced by binding to its specific receptor.
  • inflammatory and immune responses are important host defense responses, but can sometimes cause acute or chronic disease.
  • a cause that induces an inflammatory or immune response is added to a tissue, first, inflammatory cells such as neutrophils, granulocytes, lymphocytes, or macrophages or immunocompetent cells adhere to vascular endothelial cells, It moves out and accumulates in invaded or damaged tissues or in the presence of antigen.
  • chemokines As a substance that induces such a series of cell migration reactions, there is a group of chemotactic sites, so-called chemokines.
  • Chemokines are a group of site forces that induce a chemotactic reaction (chemotactic reaction), and are closely related structurally to each other due to similarities in amino acid sequences. To date, more than 30 chemokines have been reported in humans. Chemokines are largely ⁇ - or CXC-type (two cysteines separated by ⁇ amino acids) and i8 or CC from the arrangement of the first two of the four conserved cysteine residues in common. Type (two cysteines are adjacent to each other). As a CXC type chemokine, 1 L-8, ⁇ -TG.
  • CXC-type chemokines mainly induce neutrophil activation and migration.
  • CC type As human chemokines, MIP-1 ⁇ , .-1 ⁇ . RANTES, CP-K MCP-2, CP-3, U-309, and Eotaxin are known in humans.
  • CC-type chemokines mainly induce the activation and migration of monocytochrome Nomac phage.
  • B lymphocytes When B lymphocytes are infected with Epstain-Barr virus or CD4-positive T cells are infected with human herpes virus (Human Herpes virus) 6 or 7, they are selectively induced in lymphocytes whose expression is strongly induced.
  • human herpes virus Human Herpes virus 6 or 7
  • EBI-1 Epstain-Barr Virus-lnduced gene 1
  • the expressed G protein-coupled seven-transmembrane receptor EBI-1 (Epstain-Barr Virus-lnduced gene 1) is known (Birkenbach et al., J. Virol. 67: 2209). -2220, 1993; Hasegawa et al., J. Virol. 68: 5326- 5329, 1994).
  • the ligand for this receptor was unknown.
  • Induction of EBI-1 expression in host lymphocytes following infection with various lymphophilic viruses is important for acute infection, latent infection or reactivation of those lymphophilic viruses. It is considered to play a role. Therefore, it is expected that finding a ligand that specifically binds to the EBI-1 receptor will provide a therapeutic means for lymphophilic virus infection.
  • the present inventors have discovered the existence of a sequence of a DNA fragment thought to encode a new CC-type chemokine by a unique method, and complementarily cover the full-length cDNA from mRNA derived from human fetal lung tissue.
  • the various cDNA clones were separated and their full-length nucleotide sequences were determined.
  • This gene is constitutively expressed mainly in the immune system tissues such as lymph nodes, cecum, thymus, and spleen, and its protein produced by genetic engineering technology has cell-migrating activity on human T cells. was found.
  • the protein is known to be a ligand that specifically binds to the above-mentioned EB1-1, a so-called unique fan receptor, a ligand of which was not previously known.
  • the present invention has been clarified, and the present invention has been completed.
  • This new human CC-type chemokine was named ELC ( ⁇ -1-Ligand Chemokine).
  • ELC ⁇ -1-Ligand Chemokine
  • the present invention relates to a human CC-type chemokine or a mutant thereof, or a mutant thereof, which is constitutively expressed in an immune system tissue which is a ligand of the receptor EB-1 which is selectively expressed in lymphoid cells.
  • a protein which is a fragment, preferably a human CC type chemokine having an amino acid residue of amino acid residues 1 to 98 of SEQ ID NO: 1, more preferably amino acid residues 22 to 98 of SEQ ID NO: 1, or a sequence thereof Has at least one sequence selected from substitution, deletion, insertion and addition of one or several amino acid residues and has substantially the same function or activity as the human CC-type chemokine.
  • a method of performing A sample presumed to contain a gonist, an inverse gonist, or an angelic gonist is added to a binding reaction system between the protein and a specific receptor of the protein, and the inhibition of the binding is measured. Reacting directly with a specific receptor and measuring the binding and / or reactivity to that receptor.
  • FIG. 1 shows the nucleotide sequence and deduced amino acid sequence of human ELC cDNA.
  • FIG. 2 shows the results of comparing the amino acid sequences of the ELC protein of the present invention with nine kinds of known human CC-type chemokines.
  • FIG. 3 is a photograph instead of a drawing showing the result of Northern blot analysis of ELC mRNA expression in various human tissues.
  • FIG. 4 shows the genetic map of the recombinant vector pDREF-SEAP (His) 6 and the insertion position of the ELC cDNA.
  • FIG. 5 is a graph showing induction of the chemotaxis reaction of human T cell line HUT78 cells by the culture supernatant of 293 / EBNA-1 cells transfected with a vector expressing the recombinant ELC protein. As a control, the culture supernatant of 293 / EBNA-1 cells transfected with only one vector is used.
  • FIG. 6 is a graph showing the results of examining the binding of ELC-SEAP (His) 6 fusion protein using 293 / EBNA-1 cells expressing various cloned chemokine receptors. Detailed description of the invention
  • the ELC found by the present inventors is presumed to be a protein consisting of 98 amino acids from the open reading frame (0RF) predicted from the nucleotide sequence of cDNA.
  • the signal sequence is cleaved between the 2nd serine and the 2nd dalysin, and is estimated to be a basic protein of 77 amino acids with a molecular weight of about 8.8 kDa (see Figure 1).
  • Mature ELCs show significant homology to known CC chemokines, especially in CC chemokines. All four stored cysteines are stored. However, the homology with existing chemokines is about 31% even for the highest MlP-13.
  • one embodiment of the present invention is that it is constitutively expressed in immune system tissues such as lymph nodes, cecum, thymus, spleen, etc., exhibits chemotactic activity on T cells, and is selectively expressed on lymphocytes.
  • the present invention relates to a protein which is a human CC-type chemokine or a mutant thereof or a fragment thereof, which specifically binds to the human fan receptor EBI-1.
  • the present invention provides a human CC-type chemokine (ELC precursor) having the amino acid sequence of amino acid residues 1 to 98 of SEQ ID NO: 1 or the amino acid residues 22 to 98 of SEQ ID NO: 1.
  • a human CC-type chemokine having the amino acid sequence of SEQ ID NO: 1, or a sequence comprising at least one selected from substitution, deletion, insertion, and addition of one or several amino acid residues in this sequence.
  • the present invention relates to a protein which is a mutant of the human CC type chemokine or a fragment thereof.
  • CC-type chemokine variant has a sequence containing at least one selected from substitution, deletion, insertion and addition of amino acids or amino acid sequences in the amino acid sequence of the original protein. And / or a modified protein that can contain a chemically or biochemically modified or natural or unnatural amino acid, and whose function or activity is substantially the same as the CC-type chemokine. A protein that functions as an antagonist of the CC-type chemokine.
  • a protein that is a ⁇ fragment '' of the CC-type chemokine of the present invention or a mutant thereof refers to a protein having an amino acid sequence of 76 to 5, preferably 50 to 10, or about 9 amino acid residues. means.
  • the present invention relates to a polynucleotide molecule encoding the CC-type chemokine of the present invention or a mutant or a fragment thereof.
  • a polynucleotide molecule comprising:
  • a polynucleotide molecule of the present invention can be DNA or RNA.
  • DNA it may be cDNA, genomic DNA or synthetic DNA.
  • RNA in both cases of DNA and RNA, it can take a double-stranded or single-stranded form.
  • a single strand it may be a coding strand or a non-coding strand.
  • the present invention relates to variants of these polynucleotide molecules by base substitution, base addition or allelic mutation.
  • a variant by base substitution or base addition refers to the use of a different genetic code from the base sequence described in SEQ ID NO: 1 and, consequently, the amino acids 1 to 98 described in SEQ ID NO: 1. It means a mutant that can encode the same protein as the protein or the same protein as the amino acid sequence 22 to 98 described in SEQ ID NO: 1.
  • mutant due to allelic mutation means a naturally occurring base mutation based on individual or ethnic differences, and the amino acid sequence to be encoded may be changed.
  • the present invention further provides a ligated nucleotide molecule having a sequence complementary to a part of the nucleotide sequence from C at position 1 to G at position 687 of SEQ ID NO: 1, or base substitution, base addition, base modification,
  • the present invention relates to a molecule which is a mutant due to an allelic mutation and inhibits the activity or function of the protein of the present invention.
  • a sequence complementary to the 5 'non-coding portion is preferred, and more preferably a sequence complementary to the transcription initiation site, translation initiation site, 5' untranslated region, boundary region between exon and intron, or 5 'CAP region. It is a simple array.
  • Preferred lengths are from about 10 base pairs (bp) to about 60 bp.
  • the present invention relates to a vector containing the polynucleotide molecule of the present invention.
  • the vectors of the present invention include vectors having various uses, such as expression vectors, cloning vectors, and therapeutic vectors.
  • the expression vector can be used for mass production of the protein of the present invention. Details of the expression vector are shown in the following section.
  • Therapeutic vector is used in a method for introducing the polynucleotide molecule of the present invention into cells.
  • a method for introducing the polynucleotide molecule of the present invention into cells examples include a method using a viral vector and other methods (Nikkei Science, April 1994, pp. 20-45; Monthly Pharmaceutical Affairs, 36 (1) 23-48 (1994); Experimental Medicine Special Edition, 12 (15), (1994)), and the methods described in these references.
  • the polynucleotide molecule of the present invention is used for an RNA virus such as a retrovirus, an adenovirus, an adeno-associated virus, a herpes virus, a vaccinia virus, a box virus, a polio virus, and a simbis virus.
  • an RNA virus such as a retrovirus, an adenovirus, an adeno-associated virus, a herpes virus, a vaccinia virus, a box virus, a polio virus, and a simbis virus.
  • a retrovirus, an adenovirus, an adeno-associated virus, a vaccinia virus and the like is particularly preferable.
  • DNA method direct injection of plasmid into the muscle
  • ribosome method lipofectin method
  • microinjection method calcium phosphate method
  • electoral poration method DNA vaccine method and ribosome method are preferred.
  • the present invention relates to a transformant containing the above-described various vectors of the present invention.
  • the present invention also provides a transformant obtained by introducing the expression vector of the present invention into a host cell; culturing the transformant and collecting the produced protein to produce the protein of the present invention. About the method.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the protein of the present invention or a polynucleotide molecule encoding the same, or the therapeutic vector of the present invention.
  • the pharmaceutical compositions of the present invention include, for example, anti-inflammatory agents, immune response modulators, anti-infective agents.
  • polynucleotide molecule of the present invention In order for the polynucleotide molecule of the present invention to act as a medicine, an in vivo method of directly introducing it into the body, or ex vivo, using a certain cell from human, introducing a gene into the cell, and returning the cell to the body. There is a vo way.
  • the dose of the protein or polynucleotide molecule of the present invention can be appropriately adjusted depending on the age, body weight, etc. of the patient.
  • the dose is 0.001 mg to 100 mg, and is preferably administered once every several days to several months. Since the protein of the present invention is an active substance in a living body, the amount of activity of the protein, that is, the amount of the pharmaceutical composition containing the protein of the present invention, is reduced. Therefore, it is easily presumed that acute toxicity is not a problem.
  • the present invention relates to an antibody against the protein of the present invention or a mutant thereof, particularly a monoclonal antibody, and a hybridoma cell producing the monoclonal antibody.
  • the present invention provides the protein of the present invention and its specificity. By providing a relationship with a specific receptor, the process of searching for and evaluating a substance that acts as an agonist, inverse agonist or antagonist for the biological action caused by the binding of the protein of the present invention to its specific receptor. Regarding the method of inclusion. That is, a sample presumed to contain the agonist, inverse agonist or antagonist is added to the binding reaction between the protein and its specific receptor, and the inhibition of the binding is measured. And measuring the direct binding and / or reactivity to a specific receptor.
  • the present invention relates to human CC-type chemokines that are constitutively expressed mainly in immune system tissues such as lymph nodes, cecum, thymus, and spleen.
  • the sequence of this DNA fragment can be obtained, for example, from cDNA obtained from human lymph nodes, cecum, thymus, spleen, or fetal lung tissue. Primers are required to clone the cDNA to be cloned.
  • GenBank a nucleic acid sequence database published by NCBI of the United States.
  • the database includes an Expressed Sequence Tag (EST) database consisting of cDNA partial sequences, and various human CC-type chemokine amino acid sequences.
  • EST Expressed Sequence Tag
  • a search yields a partial cDNA sequence that is thought to encode a protein that has significant homology to the cc-type chemokines but differs from known chemokines.
  • a primer pair for polymerase chain reaction (PCR) is synthesized based on the obtained cDNA partial sequence.
  • RNA was extracted using a Quickprep Micro mRNA purification kit (Pharmacia), and a marathon cDNA amplification kit (CI on tech) was extracted from the poly (A) + RNA.
  • cDNA is amplified from the upstream 5 'primer to the downstream 3' end to the 3 'end of the cDNA, and the resulting cDNA fragment is converted to an appropriate base sequencing vector such as pGEM-T (Promega And pBluescript
  • the recombinant plasmid was recovered, and the nucleotide sequence of the cloned cDNA fragment was analyzed by, for example, the Sanger method (Sanger et a, Proc. Nat I. Acad. Sc, USA, 74: 5463-5467, 1977). decide.
  • the cDNA is amplified from the downstream 3 'primer to the upstream 5' end to the 5 'end of the cDNA.
  • the obtained cDNA fragment is inserted into an appropriate nucleotide sequence determination vector in the same manner as described above, the recombinant plasmid is recovered, and the nucleotide sequence of the cloned cDNA is determined.
  • the nucleotide sequence corresponding to the full-length cDNA is determined.
  • the obtained cDNA encoding the ELC protein is inserted into an appropriate expression vector to prepare an expression vector for expressing the ELC protein.
  • suitable expression vectors include, for example, pRSET, pGEEX.pKK233-2 for bacteria, pYES2 for yeast, pVL1393 for insect cells, pEF-B0S, pSRa, pDR2 for animal cells, etc. Respectively.
  • Transformants are prepared by introducing this expression vector into an appropriate host cell, for example, a bacterium, yeast, insect cell or animal cell.
  • ⁇ Prokaryotic microorganisms such as Escherichia coli include signal sequences derived from secreted proteins of prokaryotic microorganisms (eg, It can be expressed as a fusion protein in which the signal peptide (OMPa) and the mature ELC protein are fused under the control of a strong promoter (eg, T7 promoter).
  • OMPa signal peptide
  • yeast it can be expressed as a fusion protein in which a signal sequence derived from a natural precursor of a yeast secretory protein (eg, a pheromone ⁇ prebuilt opening sequence) and an thermogenic ELC protein are fused.
  • the gene for the precursor protein of the ELC protein which contains a signal sequence that already exists, is inserted downstream of a strong promoter (eg, EF-1 ⁇ promoter) to form an effective selection marker (eg, dihydrogen). It can be introduced into animal cells (for example, CHO dhfr-cells) together with folate reductase), and cells can be selected based on resistance to a drug (in this case, methotrexate) to establish a highly expressing cell line.
  • the gene can be expressed by incorporating the gene for the ELC protein precursor containing the signal sequence into a virus or retrovirus, and infecting animal cells with the recombinant virus. By culturing these transformants, ELC protein can be produced and secreted.
  • the mature ELC protein or a protein fragment thereof can be obtained by the method described in the literature based on the determined base sequence (for example, using a solid phase method), paying attention to the presence of two disulfide bonds.
  • Total synthesis can be performed by Clark-Lewis et al., Biochemistry 30: 3128-3135, 1991) or by using a conventional peptide synthesizer.
  • Purification of the obtained protein can be performed by a method known to those skilled in the art, for example, by a combination of affinity chromatography, ion exchange chromatography, gel filtration chromatography, reverse phase chromatography, and hydrophobic chromatography.
  • Mutants and fragment proteins of the ELC protein of the present invention can be mutated by gene recombination techniques well known to those skilled in the art (Sambrook eta I., Molecular Cloning: AI aboraroy manual, 2nd edn.New York, Cold Spring Harbor Laboratory). It can be prepared using DNA into which is introduced.
  • Antibodies against the ELC protein of the present invention include, for example, a synthetic peptide synthesized with a conventional peptide synthesizer based on a part of the deduced ELC amino acid sequence, a bacterium, and a yeast transformed with an ELC-expressing vector.
  • ELC proteins produced by insect cells, animal cells, animal cells, etc. are purified by ordinary protein chemical methods, and these are used as immunogens to immunize animals such as mice, rats, hamsters, egrets, Prepare an antibody of origin (polyclonal antibody).
  • lymphocytes are removed from the spleen or lymph nodes of immunized mouse rats and fused with myeloma cells, and the method of Kohler and Mi Istein [Nature, 256, 495-497 (1975)] or an improved method thereof.
  • a hybridoma can be prepared according to the method of Ueda et al. [Pro Na 11. Acad. Sc, USA, 79: 4386-4390, 1982), and a monoclonal antibody can be produced from the hybridoma.
  • a monoclonal antibody of the ELC protein can be obtained by the following steps.
  • the presence of the mRNA and protein of the ELC of the present invention can be carried out using a detection method for ordinary specific mRNA and protein (Sambrook et al., Molecular Cloning: AI abo roy manual, 2nd edn. New York, Cold Sring Harbor Laboratory 1989; Harlow and Lane, Antibodies: A laboratory manual, New York, Cold Spring Harbor Laboratory 1988).
  • mRNA can be detected by Northern blot analysis using antisense RNA or cDNA as a probe or in situ hybridization method.
  • the mRNA can be converted to cDNA using reverse transcriptase, and then detected by polymerase chain reaction (PCR) using an appropriate combination of primers.
  • the protein can be detected by immunoprecipitation using an antibody specific to the ELC protein, Western blot, or the like.
  • ELC labeled with radioisotope an enzyme such as peroxidase or alkaline phosphatase, or a fluorescent dye
  • unlabeled ELC with a known concentration unlabeled ELC with a known concentration
  • anti-ELC polyclonal antibody derived from serum or monoclonal antibody An antigen-antibody competition reaction is performed by adding one null antibody. After appropriately changing the concentration of the unlabeled antigen, the labeled antigen bound to the antibody and the labeled antigen not bound to the antibody are separated by an appropriate method, and the radioactivity and enzyme activity of the labeled antigen bound to the antibody are separated. Or measure the fluorescence intensity.
  • the amount of unlabeled antigen increases, the amount of labeled antigen that binds to the antibody decreases. This relationship is graphed to obtain a standard curve.
  • one of the two types of monoclonal antibodies that recognize different epitopes on the ELC protein is immobilized, and the other is labeled by one of the above methods, and the amount of ELC bound to the immobilized antibody is determined by the amount of the labeled antibody.
  • sandwich method of detecting and quantifying by means of is also possible.
  • the chemokine activity of the ELC protein of the present invention can be determined, for example, by placing ELC on one side of a culture vessel partitioned with a filter having a certain diameter in a test tube and placing target cells on the other side. After a certain period of time, the number of cells that have moved through the pores of the filter to the side where ELCs are present can be compared with the number of random movements. In vivo, it can also be demonstrated by administering purified ELC protein to animals and detecting cell invasion and aggregation by histological methods.
  • the Expressed Sequence Tag (EST) database which is a part of the nucleic acid sequence database GenBank released by NCBI in the United States and composed of partial sequences derived from various cDNAs, is used for amino acids of various human CC-type chemokines. A search was performed using TBLASTN search software based on the acid sequence. Seven EST data (GenBank data) that have significant homology to CC-type chemokines but are thought to encode chemokine proteins different from known chemokines Session No .: T97490, D31180, D3143K W05519, W0740K N71167, N80273).
  • ELC mRNA was detected by polymerase chain reaction (PCR) using a combination of primers specific to ELC.
  • PCR polymerase chain reaction
  • a QuickPrep Micro mRNA purification kit (Pharmacia) ) was used to extract mRNA.
  • mRNA was synthesized using a Preamplification System (GIBCO-BRL).
  • PCR was performed using Ampl iTaq Kit (Takara Shuzo). That is, the obtained single-stranded cDNA was used as type III, and 400 nM of each of the nfJCC-6F primer, nNCC-6R primer and 100 U / ml Amp I iTaq DNA polymerase I were added to a reaction buffer (10 mM Tris-HC). PCR was performed using a DNA Thermal Cycler (Perkin-EImer) in addition to pH 8.3, 50 mM KCL, 1.5 mM MgCI2, 0.1 gelatin, 200 M each of dATP, dGTP, dCTP, and dTTP.
  • a DNA Thermal Cycler Perkin-EImer
  • the reaction was pre-treated at 94 ° C for 1 minute, followed by 5 reaction cycles of 94 ° C for 30 seconds, 72 ° C for 2 minutes, 5 cycles of 94 ° C for 30 seconds, and 70 ° C for 2 minutes. Five cycles, and finally 25 cycles of 94 ° C for 30 seconds and 68 ° C for 2 minutes were performed.
  • the amplified DNA fragment was separated by agarose electrophoresis and stained by ethidium bromide method. Approximately 170 bp of DNA derived from ELC cDNA was detected, confirming that human fetal lung tissue expresses ELC mRNA.
  • cDNA is synthesized from mRNA derived from human fetal lung tissue using the Marathon cDNA Amplification Kit (Clontech). That is, a 5 / I aqueous solution containing human fetal lung mRNA 1 and Marathon cDNA synthesis primer 10 pmole was heated at 70 ° C for 2 minutes, cooled on ice, and added to dATP, dCTP, dGTP and dTTP ( each 1 mM) and MMLV the (Moloney Murine Leukemia Virus) reverse transcriptase (100 units) was added, 50 mM Tr i s-HCI (pH8.3), 6 mM MgCI 2, 75 mM reaction solution 10 I in KCI The single-stranded cDNA synthesis reaction was performed at 42 ° C for 4 hours.
  • T4 DNA polymerase (10 units) was added to the reaction solution, and the mixture was reacted at 16 ° C for 45 minutes to blunt the cDNA. After the reaction, phenol extraction and ethanol precipitation were performed, and the DNA was dissolved in 10 I of distilled water.
  • Marathon cDNA Adaputa one 20 pmo le and T4 DNA Riga Ichize the (1 Yuni' g) was added to 5 il of solvent solution of them, 50 mM Tr i s-HCI (pH7.8) v 10 mM MgCI 2, 1 mM DTT, 1 mM ATP.
  • a RACE reaction (Frohman et al., Proc. Nat I. Acad. Sci. USA 85: 8998-9002, 1988) was performed.
  • 5 A of the denatured cDNA was added to dATP, dCTP, dGTP, dTTP (0.2 mM each), TAKARA LA Taq (2.5 units), and TaqStart antibody (0.55 ⁇ L).
  • g add 10 pmo Ie of API primer that binds to a part of the adapter, and 10 pmole of nNCC-6R primer (SEQ ID NO: 3), and in a 1x TAKARA LA Taq buffer reaction mixture at 94 ° C in 50 ⁇ I.
  • PCR was carried out for 30 cycles at 94, 30 seconds; 60 ° C, 30 seconds; 68 ° C, 4 minutes.
  • the 3 ′ RACE reaction was performed under the same reaction conditions as above, except that the nNCC-6F primer (SEQ ID NO: 2) was used instead of the nNCC-6R primer.
  • each PCR product was separated by 23 ⁇ 4 low melting point agarose gel electrophoresis, and the main 5 'RACE fragment (about 670 bp) and the main 3' RACE fragment
  • Plasmid DNA was extracted from several of the colonies obtained in the above steps, and the nucleotide sequences at the 5 'and 3' ends of the cDNA were examined using SP6 motor, primer and T7 promoter and primer. However, all had almost the same nucleotide sequence as EST W07401. Therefore, clones obtained from each of the 5 'RACE reaction and the 3' RACE reaction were selected one by one (hereinafter referred to as 5'-RACE cDNA and 3'-RACE cDNA), and the entire nucleotide sequence of them was determined by Sanger et al. Determined according to the method (Proc. Nat I. Acad. Sci. USA 74: 5463-5467, 1977).
  • the full-length ELC cDNA was determined from the two partially overlapping cDNA sequences thus obtained. As a result, it was found that there was a nucleotide sequence encoding a protein consisting of 98 amino acid residues including methinenin defined by the translation initiation codon ATG, which appears first.
  • the amino acid sequence of this protein is not identical to known chemokines, but has significant homology and contains four conserved cysteine residues that are structural features of chemokines. The presence of a highly hydrophobic signal sequence-like sequence on the N-terminal side, which suggests a novel chemokine.
  • FIG. 1 shows the determined nucleotide sequence of the full-length cDNA and the amino acid sequence of the longest translation frame (open read frame: 0RF) starting from the predicted start codon.
  • This gene has 0RF consisting of 98 amino acids, and has about 20 strongly hydrophobic amino acid sequences at the N-terminus, which are presumed to be signal peptides characteristic of secreted proteins. The molecular weight of this 98 amino acid protein was 10,992.
  • the calculated cleavage site for the fernal peptide was estimated to be between serine at position 21 and dalysin at position 22.
  • the putative mature protein consisting of 77 amino acid residues after signal peptide cleavage is presumed to be a secreted protein, with a molecular weight of 8,779 and an isoelectric point of 10.1. It was 6.
  • ELC niRNA expression analysis of ELC niRNA by Northern blot analysis 2 g of poly (A) + RNA isolated from various human tissues was subjected to agarose gel electrophoresis and transferred to a nylon membrane (multiple tissue plot). A hybridization reaction was performed using 5P-RACE cDNA of ELC, which was purchased from a company and labeled with 32 P by a multi-prime DNA labeling system (manufactured by Stratagene), as a probe. The hybridization solution was prepared by adding 100 ⁇ g / ml of salmon sperm DNA to QuikHyb (manufactured by Strat agene) and hybridization was performed at 65 ° C. for 1 hour.
  • FIG. 3 shows the expression of ELC mRNA in various human tissues.
  • the results in FIG. 3 revealed that mRMA of ELC was strongly and constitutively expressed in immune system tissues, particularly in lymph nodes, cecum, thymus, spleen, and the like.
  • the ELC protein was produced in animal cells by introducing cDNA encoding the ELC protein.
  • the cDNA encoding the full-length ELC protein was designated as ELC 5'-RACE cDNA (above), and the 5'-SALT ELC primer (SEQ ID NO: 4) and the 3'-ELC-Xbal primer (SEQ ID NO: 4) were used.
  • ELC 5'-RACE cDNA above
  • SEQ ID NO: 4 the 5'-SALT ELC primer
  • 3'-ELC-Xbal primer SEQ ID NO:
  • the obtained PCR product was digested with the restriction enzymes Sal I and Xbal, and the digestion was performed between the Sail and Xbal sites of the expression vector pDREF-Hyg (Imai et al., J. Biol. Chem. 271: 21514-21521, 1996).
  • pDREF-ELC which expresses the ELC protein.
  • This vector was introduced into 293 / EBNA-1 cells (Invitrogen) using Lipofectamine (Gibco-BRL).
  • As a control only the vector pDREF-Hyg was introduced. After 3 to 4 days of culture, the culture supernatant was collected, sterilized by filtration with a 0.22 ⁇ m filter, and the cell migration activity was measured as shown in the following test examples.
  • the carboxyl terminus of the ELC is A fusion protein in which phosphatase (SEAP) and histidine tag (His) 6 were fused was prepared.
  • SEAP can be detected and quantified by an immunological method using an enzyme activity anti-SEAP antibody.
  • Histidine tag (His) s having six consecutive amino acid histidines is introduced into Uanknecht et al., Proc. Natl. Acad. Sc, USA 88, which is introduced for affinity purification of a fusion protein using a nickel affinity column. : 8972-8976, 1991).
  • FIG. 5 shows a schematic diagram of a vector pDREF-SEAP (His) s for expressing a fusion protein of ELC and SEAP-(His)-.
  • Preparation of pDREF-SEAP (His) 6 was performed as follows. Using the plasmid pSEAP-Enhancer manufactured by CI ont ech as type II, a DNA encoding the amino acid sequence obtained by adding 6 histidines (His) to SEAP to a 5'-Xbal-AP primer (sequence number
  • the obtained DNA is digested with restriction enzymes Xbal and Notl, and then introduced between XbaI and No11 site of pDREF-Hyg (Imai et al., J. Biol. Chem. 271: 21514-21521, 1996).
  • pDREF-SEAP His
  • 0RF of the ELC cDNA was inserted between the SalI and Xbal sites of the pDREF-SEPAP (His) 6 vector, and the ELC was composed of 5 amino acid linkers (Se r -A vector pDREF-ELC_SEPAP (His) s encoding a protein fused to SEAP- (His) 6 via Arg- Ser_Ser-Gly) was prepared.
  • a cDNA encoding the full-length 0RF of the ELC was prepared by combining the 5'-RACE cDNA (above) of the ELC with the 5'-Salt ELC primer (SEQ ID NO: 4) and the 3 ' -Amplification was performed by PCR using ELC-Xba I primer 1 (SEQ ID NO: 5).
  • the pDREF-ELC-SEAP (HIS) 6 vector was introduced into 293 / EBNA-1 cells (Invitrogen) using ribofectamine (GIBC0-BRL). After 3 to 4 days of culture, collect the culture supernatant, pass through a 0.22 mm pore size filter, and mix with 20 mM HEPES (H 7.4).
  • the sample is diluted with a culture solution (D-MEM containing 10% fetal serum), added to the microplate, reacted for 1 hour at room temperature, washed with a washing solution (PBS containing 0.02% Tween-20), A 1-fold diluted bivalent rabbit heron anti-PLAP antibody was added and reacted for 1 hour. After further washing, peroxidase-conjugated streptavidin (Vector) was added and reacted for 30 minutes. After washing, the activity of the bound peroxidase was detected with 3.3'-5,5'-tetramethylbenzidine. The reaction was stopped with 1 NH 2 SO 4 and the absorbance at 450 nm was measured.
  • D-MEM containing 10% fetal serum
  • alkaline phosphatase The activity of alkaline phosphatase (AP) was measured by a chemiluminescence method using Great EscApe Detection Kit (manufactured by CI on tech) and determined as relative light intensity / second (RLU / s). The preparation of an AP standard curve was performed using purified placenta-type ALPHA rifatatases (manufactured by Cosmo Corporation). SEAP and ELC-SEAP actually used in the test were
  • the human T cell line HUT78 was used as the cells (Itnai et al., J. Biol. Chem. 271: 21514-21521, 1996).
  • the test solution was diluted with a culture solution (RPMI-1640, 20 mM Hepes (pH 7.4), UBSA) and added to the lower well of a 48-well chemotaxis chamber (chemotaxis chamber, manufactured by Neuro Probe). In the upper well, suspended in the above culture solution
  • ELC-SEAP His 6 was performed as follows to determine the N-terminus of mature secreted ELC. Add 20 mM Tri S-HCI (pH 8.0) and 1 OmM imidazole to the culture supernatant filtrate (containing 20 mM HEPES (pH 7.4) and 0.025! Sodium azide) obtained in Example 4, and add A buffer. The solution was applied to a 1 mI nickel affinity column Ni-NTA-Agarose (manufactured by QIAGEN) equilibrated with a liquid (20 mM Tris-HCI (pH 8.0) / 1 OmM imidazole).
  • the column to which the ELC-SEAP (His) 6 fusion protein was bound was washed with B buffer (20 litter Tris-HCI (pH 8.0), ⁇ 0 mM imidazole, 150 mM NaCI), and then buffer C (20 m Tris-HCI (pH 8) .0), 100 mM imidazole, 150 mM NaCl).
  • B buffer (20 litter Tris-HCI (pH 8.0), ⁇ 0 mM imidazole, 150 mM NaCI
  • buffer C (20 m Tris-HCI (pH 8) .0), 100 mM imidazole, 150 mM NaCl.
  • the fractions containing the ELC-SEAP (His) 6 fusion protein were identified using SDS-PAGE.
  • the N-terminal amino acid sequence of this purified ELC-SEAP (His) 6 fusion protein was determined using an amino acid sequencer (manufactured by Shimadzu) and analyzed using Gly-Thr-Asn-Asp-Ala-Glu-Asp. It was hot.
  • This amino acid sequence contains a signal between the serine residue at position 2 2 and the glycine residue at position 22 which are the cleavage sites of the deduced signal sequence in the amino acid sequence deduced from the nucleotide sequence shown in Fig. 1.
  • the peptide was completely cleaved to completely match the N-terminal amino acid sequence expected when a mature secreted ELC protein consisting of 77 amino acids was obtained.
  • the chemokine receptor 8 species already cloned and reported that is, CCR1 (Neote et al., Cell 72: 415) -425, ⁇ 993), CCR2 (Charo et a, Pro Natl. Acad. Sci. USA 91: 2752-2756, 1994), CCR3 (Ki taura et al., J. Bio Chem. 271: 7725-) 7730, 1996), CCR4 (Power et al., J. Biol. Chem. 270: 19495-19500, 1995), CCR5 (Kitaura et al., J. Biol. Chem.
  • Cells expressing each chemokine receptor were prepared as follows. First, cDNAs corresponding to ORFs of eight chemokine receptors were inserted between Xba I and No. 11 site of pDREF-Hyg vector (Imai et al., J. Biol. Chem.
  • each of the expression vectors was introduced into 293 / EBNA-1 cells derived from human fetal kidney (Invitrogen) using lipofectamine (Gibco-BRL) and expressed. After 2 days of culture, the cells were collected, suspended in RPM 1640 containing 20 mM HEPES (pH 7.4),]% BSA, 0.02 sodium azide, and ⁇ ⁇ M of ELC-SEAP (His) 6 and 4 ⁇ 10 5 The cells were reacted for 1 hour at room temperature in a solution of 200 ⁇ I.
  • ELC-SEAP (His) 6 is a novel receptor for which no specific ligand has been known so far--EB 1 (Birkenbach et al., J. Virol. 67: 2209-2220, 1993).
  • Chemokines which induce leukocyte migration and infiltration into tissues, are essential substances for inflammatory and immune responses in vivo.
  • CXC type and CC type are mainly known as chemokines, and there are multiple types of each type, ranging from producing tissues, producing cells, types of stimuli to induce production, induction of production to production cessation They exhibit different properties with respect to reaction time, types of target cells that induce migration, and the presence of specific receptors.
  • ELC specifically binds to G protein-coupled seven-transmembrane receptor EB1 (Birkenbach et al., J. Virol. 67: 2209-2220, 1993), which is selectively expressed on lymphocytes.
  • G protein-coupled seven-transmembrane receptor EB1 By exhibiting chemotactic activity on T cells, it is easily expected that they will participate in acute or chronic inflammatory and immune responses involving lymphocytes. Therefore, the ELC of the present invention, through further elucidation of its functions, understands the inflammatory and immune reactions involving lymphocytes and provides a new means to induce or suppress such phenomena. Offer.
  • the ELC of the present invention is constitutively expressed at a considerable level in lymphoid tissues such as lymph nodes, cecum, thymus, spleen, etc. And homing in lymphoid tissues, and is expected to be involved in lymphocyte migration and settlement, maturation and differentiation, antigen recognition, survival, proliferation, etc. in lymphoid tissues. Therefore, the ELC of the present invention, by elucidating its function, is useful for understanding lymphocyte migration and settlement in various lymphoid tissues, differentiation and maturation, antigen recognition, regulation of cell growth and survival, etc. It provides useful tools to control such phenomena.
  • the ELC protein or a mutant thereof provided by the present invention regulates a physiological or pathological biological reaction involving ELC by enhancing or suppressing the action of ELC in vivo. It is possible to do.
  • the specific binding between ELC and EB1 provided by the present invention is selectively expressed on lymphocytes, and is expressed by Epstein-Barr virus, Human Herpesvirus 6, Human Herpesvirus7. It provides a new means to elucidate the physiological and pathological functions of EBI-1, whose expression in lymphocytes is strongly induced by infection with lymphophilic herpes virus such as E. coli.
  • Antagonists and agonists against EBI-1 to block the specific binding of ELC to EB-1 and to promote or suppress the biological effects caused by the binding of ELC to EB-1 It provides a means for searching and evaluating.
  • Angiogonist agonists and the like for the binding reaction between ELC and EB-1 can regulate physiological or pathological biological reactions involving ELC and EBI-1.
  • induction of EBI-1 expression in host lymphocytes following infection with various lymphophilic viruses plays an important role in the acute, latent, or reactivation of those lymphophilic viruses. Therefore, agonists, inverse agonists or antagonists to ELC protein or its variants, or EBI-1 are expected to show therapeutic effects against lymphophilic herpesvirus infection Is done.
  • the polynucleotide (DNA or RNA, double-stranded or single-stranded) encoding the ELC provided by the present invention in full length or in part can be directly administered to a living body as a polynucleotide by an appropriate method.
  • the cells transformed cells
  • the cells can be returned to the body by introducing them into cells outside the body, or they can be introduced directly into a suitable vector and introduced into the body.
  • the nucleotide sequence of ELC is useful for detecting and analyzing ELC gene mutation. It is also useful for specifically detecting and quantifying ELC gene expression (mRNA) and protein expression. This provides a new means for diagnosis and investigation of the causes of blood system diseases, immune system diseases, infectious diseases, cancers, etc. involving the ELC gene and ELC protein, and new means for diagnosis and treatment of such diseases. It is expected to provide.
  • Sequence type nucleic acid
  • Organism name human
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid

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Abstract

L'invention concerne une nouvelle chémokine CC humaine ELC ainsi qu'une molécule polynucléotidique la codant. La protéine précitée est une chémokine CC humaine exprimée de manière constitutive dans des tissus lymphoïdes et elle est un ligand du récepteur EBI-1 connu comme subissant une expression sélective dans des lymphocites ou un fragment ou un variant de ceux-ci. L'invention concerne également une liaison spécifique de cette protéine au récepteur transmembranaire-7 EBI-1 conjugué à une protéine G ainsi qu'une méthode de dosage d'agonistes et d'antagonistes de ce système de ligand/récepteur spécifiques au moyen de cette liaison.
PCT/JP1997/004573 1996-12-13 1997-12-12 Chemokine cc humaine elc WO1998026071A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000009151A1 (fr) * 1998-08-17 2000-02-24 Schering Corporation Regulation de l'activite de cellules dendritiques

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996022374A1 (fr) * 1995-01-19 1996-07-25 Incyte Pharmaceuticals, Inc. Nouvelle chemoquine exprimee dans la rate de foetus, son mode de production et ses utilisations
WO1996024668A1 (fr) * 1995-02-08 1996-08-15 Human Genome Sciences, Inc. Chemokine humaine beta-11 et chemokine humaine alpha-1
WO1996039522A1 (fr) * 1995-06-05 1996-12-12 Human Genome Sciences, Inc. Chemokine humaine beta-11 et chemokine humaine alpha-1
WO1997015594A1 (fr) * 1995-10-24 1997-05-01 Smithkline Beecham Corporation Nouvelles chimiokines utilisees pour mobiliser des cellules souches

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996022374A1 (fr) * 1995-01-19 1996-07-25 Incyte Pharmaceuticals, Inc. Nouvelle chemoquine exprimee dans la rate de foetus, son mode de production et ses utilisations
WO1996024668A1 (fr) * 1995-02-08 1996-08-15 Human Genome Sciences, Inc. Chemokine humaine beta-11 et chemokine humaine alpha-1
WO1996039522A1 (fr) * 1995-06-05 1996-12-12 Human Genome Sciences, Inc. Chemokine humaine beta-11 et chemokine humaine alpha-1
WO1997015594A1 (fr) * 1995-10-24 1997-05-01 Smithkline Beecham Corporation Nouvelles chimiokines utilisees pour mobiliser des cellules souches

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
J. BIOL. CHEM., 272(21), (1997), YOSHIDA R. et al., "Molecular Cloning of a Novel Human CC Chemokine EBI1-Ligand Chemokine that is a Specific Functional Ligand for EBI1, CCR7", p. 13803-13809. *
J. IMMUNOL., 158(3), (1997), ROSSI D.L. et al., "Identification Through Bioinformatics of Two New Macrophage Proinflammatory Human Chemokines", p. 1033-1036. *
J. VIROL., 68(8), (1994), HASEGAWA H. et al., "Induction of G Protein-Coupled Peptide Receptor EBI 1 by Human Herpesvirus 6 and 7 Infection in CD4+ T Cells", p. 5326-5329. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000009151A1 (fr) * 1998-08-17 2000-02-24 Schering Corporation Regulation de l'activite de cellules dendritiques

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