JPH10508742A - Human chemokine polypeptide - Google Patents

Abstract

Human chemokine polypeptides and DNA (RNA) encoding such chemokine polypeptides and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such chemokine polypeptides for the treatment of leukemia, tumors, chronic infections, autoimmune disease, fibrotic disorders, wound healing and psoriasis. Antagonists against such chemokine polypeptides and their use as a therapeutic to treat rheumatoid arthritis, autoimmune and chronic inflammatory and infective diseases, allergic reactions, prostaglandin-independent fever and bone marrow failure are also disclosed.

Description

DETAILED DESCRIPTION OF THE INVENTION                         Human chemokine polypeptide   The present invention relates to newly identified polynucleotides, such polynucleotides Polypeptides, such polynucleotides and polypeptides The use of tides, and also the use of such polynucleotides and polypeptides Related to manufacturing. In particular, the polypeptides of the present invention are sometimes referred to below as "Ckβ -4 "and" Ckβ-10 "and are referred to as" chemokine polypeptides ". Human chemokine beta-4 and human chemokine beta-10 It is. The present invention also relates to inhibiting the action of such polypeptides. You.   Chemokines are the output of small secreted cytokines that are structurally and functionally related. Emerging superfamily. All chemokines are amino acid levels Show 25-75% homology at the same level, as do the polypeptides of the invention. Contains spatially conserved cysteine residues. Peptides that activate neutrophils ( The “C—X—C branch (also known as the NAP) / IL-8 family Lunch) "(depending on the position of the first two cysteines in the conserved motif) Members mainly act on neutrophils (eg, IL-8 and NAP-2). Thus, it exerts an early pro-inflammatory activity, but "CC branch (Bra Members) appear to attract certain mononuclear cells. "C-C minutes Members of the "branch" include PF4, MIP, MCP and the chemo of the present invention. Cain polypeptides are included.   Many biological activities have been attributed to this chemokine family . Macrophage inflammatory proteins 1α and 1β are expressed in different lymphocyte populations (p opulations) and chemotaxis to monocytes (Schall, TJ, Cytokine, 3). : 165 (1991)), but MCP-1 is described as a specific monocyte chemoattractant. (Matsushima, K. et al., J. Exp. Med., 169: 1485 (198) 9)). The common function of this chemokine family is that different sets of cells, such as If The ability to stimulate chemotactic migration of immune cells (leukocytes) and fibroblasts. these Chemokines can also activate certain cells in this family. Wear.   Immune cells that respond to chemokines have a vast number of in vivo functions Therefore, the regulation by such chemokines is an important area in the treatment of disease. It is.   For example, eosinophils destroy parasites and reduce parasite infections. Eosinophil It is also a cause of chronic inflammation in the respiratory tract airways. Macrophages contain spinal cord It serves to reduce tumor formation in vertebrates. In addition, basophils are allergic Releases histamine, which may play an important role in sexual inflammation. Therefore, such Promoting and inhibiting cells has broad therapeutic utility.   According to one aspect of the present invention, novel polypeptides that are Ckβ-4 and Ckβ-10 And fragments, analogs and derivatives thereof. Departure The clear polypeptide is of human origin.   According to another aspect of the invention, a polynucleotide encoding such a polypeptide Provide a tide (DNA or RNA).   According to yet a further aspect of the present invention, such polypeptides are produced by recombinant techniques. And a method for manufacturing the same.   According to a still further aspect of the invention, such a polypeptide, or such Polynucleotides encoding novel polypeptides can be used for therapeutic purposes, e.g., solid (s olid) to treat tumors, chronic infections, autoimmune diseases, psoriasis, asthma, allergies Provides a method for regulating hematopoiesis and promoting wound healing I do.   In accordance with yet a further aspect of the present invention, there are provided antibodies against such polypeptides. Offer.   According to yet another aspect of the invention, for example, autoimmune diseases, chronic inflammation and infectious diseases , Histamine-mediated allergic reaction, independent of prostaglandins Fever, bone marrow failure, silicosis, sarcoidosis, hypereosinophilia syndrome and lung flame Used to inhibit the action of such polypeptides in the treatment of disease. Antagonists / inhibitors for such polypeptides provide.   These and other aspects of the invention will be apparent to those skilled in the art from the teachings herein. Or maybe.   The following drawings illustrate aspects of the invention and are encompassed by the following claims. It is not intended to limit the scope of the invention.   FIG. 1 shows the cDNA sequence of Ckβ-4 and the corresponding deduced amino acid sequence. Since the first 24 amino acids represent the predicted leader sequence of Ckβ-4, The resulting mature polypeptide comprises 72 amino acids. Markers for amino acids Use standard one-letter abbreviations.   FIG. 2 shows the cDNA sequence of Ckβ-10 and the corresponding deduced amino acid sequence. . The first 23 amino acids represent the putative leader sequence of Ckβ-10 Thus, the deduced mature polypeptide comprises 75 amino acids. amino acid Use the standard one-letter abbreviations for   FIG. 3 shows the relationship between Ckβ-4 and the mature peptide of eotaxin (lower). Shows amino acid sequence homology.   FIG. 4 shows the amino acid sequence between Ckβ-10 (top) and human MCP-3 (bottom). Shows the homology of   According to one aspect of the present invention, a mature Ckβ-4 polypeptide having the deduced amino acid sequence of FIG. A Lipeptide or ATCC Deposit No. 75848 on July 29, 1994. Mature polypeptide encoded by the cDNA of the clone deposited in Mature Ckβ-10 polypeptide having the deduced amino acid sequence of FIG. Claw deposited as ATCC Deposit No. 75849 on July 29, 994 Isolated nucleus encoding the mature polypeptide encoded by the cDNA of the protein Provide an acid (polynucleotide).   The polynucleotide encoding Ckβ-4 is a cDNA library obtained from human gallbladder. Found throughout the library. Ckβ-4 is structurally a member of the chemokine family. Have a relationship. It is an open protein that encodes a 116 amino acid residue protein Reading frames, of which the first 24 amino acid residues are predicted. Because of the defined leader sequence, the mature protein contains 92 amino acids. I mean. The protein responds to eotaxin over the entire coding sequence, It shows the highest degree of homology with 20% identity and 37% similarity. Four spatially conserved cysteine residues in chemokines were identified as poly- It is also important to be found in peptides.   The polynucleotide encoding Ckβ-10 was obtained from 9 weeks of early human tissue. Found in the cDNA library. Ckβ-10 is structurally Family. It codes for a protein of 98 amino acid residues. Open reading frames, of which the first 23 Since the acid residue is a predicted leader sequence, the mature protein Comprising amino acids. The protein has the entire coding sequence relative to MCP-3. The highest degree of homology with 65% identity and 77% similarity Shows sex.   The polynucleotide of the invention may be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA. The DN A can be double-stranded or single-stranded, and if single-stranded, can be the coding strand or non-stranded. It can be the coding (anti-sense) strand. Coding sequence encoding the mature polypeptide The columns are the coding sequence shown in FIGS. 1 and 2 or the code of the deposited clone. Sequence, or as a result of duplication or degeneracy of the genetic code, The same mature polypeptide as the DNA of FIGS. 1 and 2 or the deposited cDNA May be a different coding sequence.   The mature polypeptide of FIG. 1 and FIG. The polynucleotide encoding the mature polypeptide to be encoded includes: Can be rare: coding sequence for mature polypeptide only; coding sequence for mature polypeptide And leader or secretory or proprotein sequences Additional coding sequence; the coding sequence for the mature polypeptide (and optionally, additional coding Do Sequences), and introns or non-coding sequences of the coding sequence of the mature polypeptide. Non-coding sequences such as 5 'and / or 3'.   Thus, the term "polynucleotide encoding a polypeptide" A polynucleotide containing only the coding sequence of the peptide, and also additional Includes polynucleotides that include coding and / or non-coding sequences.   The present invention further provides a polypeptide having the deduced amino acid sequence of FIGS. 1 and 2. Of the polypeptide encoded by the cDNA of the clone or the deposited clone. Mutations of the above polynucleotides encoding fragments, analogs and derivatives About the body. Polynucleotide variants are naturally occurring versions of a polynucleotide It may be an allelic variant or a non-naturally occurring variant of the polynucleotide.   Accordingly, the present invention includes the same mature polypeptide as shown in FIGS. Or the same mature polypeptide encoded by the cDNA of the deposited clone. Encoding polynucleotides, and also variants of such polynucleotides Variants are included, and these variants are polypeptides or variants of FIGS. 1 and 2. A fragment of the polypeptide encoded by the cDNA of the deposited clone; Encode the derivative or analog. Such nucleotide variants include deletions Variants, substitution variants and addition and insertion variants are included.   As indicated above, the polynucleotide comprises the code shown in FIG. 1 and FIG. The coding sequence of a naturally occurring allelic variant of the sequence or the deposited clone. It may have coding sequences that are naturally occurring allelic variants. Known in the industry As such, allelic variants can have one or more nucleotide substitutions, deletions, or deletions. Is another form of a polynucleotide sequence that may have additions, which are encoded It does not substantially alter the function of the polypeptide.   The invention also provides that the coding sequence for the mature polypeptide is derived from a polypeptide derived from a host cell. Polynucleotide sequences that aid in the expression and secretion of tides, such as polynucleotides from cells Same as leader sequence, which acts as a secretory sequence to control peptide trafficking Also included are polynucleotides that can be fused in reading frame. Poly with leader sequence The peptide is a preprotein and is cleaved by the host cell to form the mature form of the polypeptide. May have a leader sequence that forms a repeptide. The polynucleotide is Proproteins, which are mature proteins with additional 5 'amino acid residues, are also Can code. A mature protein having a prosequence is a proprotein; and Inactive form of the protein. Once the prosequence is cleaved, the active mature protein Quality remains.   Thus, for example, a polynucleotide of the invention may comprise a mature protein, Protein with sequence, or both prosequence and presequence (leader sequence) Can be encoded.   The polynucleotide of the present invention also allows for purification of the polypeptide of the present invention. It may also have the coding sequence fused in frame to the marker sequence. In the case of a bacterial host, Marker sequence to provide for purification of the mature polypeptide fused to the marker The hexa-histidine tag provided by the pQE-9 vector Well, or when using, for example, a mammalian host, eg, COS-7 cells Alternatively, the marker sequence may be a hemagglutinin (HA) tag. HA tag Corresponding to an epitope obtained from the influenza hemagglutinin protein ( Wilson, I. et al., Cell, 37: 767 (1984)).   The invention further provides at least 50%, and preferably 70%, identity between the sequences. A polynucleotide that hybridizes to the above sequence, if any. Book The invention particularly hybridizes to the above polynucleotides under stringent conditions. Related polynucleotides. As used herein, "stringers" The term "ant conditions" refers to at least 95%, and preferably at least Hybridization will only occur if there is also 97% identity Means that. In a preferred embodiment, the polynucleotide hybridizes to the above-described polynucleotide. The polynucleotides shown in FIGS. 1 and 2 or the deposited cDNA Substantially the same biological function or activity as the mature polypeptide encoded by A. Encodes the retained polypeptide.   The deposit referred to herein relates to the international recognition of the deposit of microorganisms in patent proceedings. Will be kept under the terms of the Plague Treaty. These deposits are simply It is provided as a convenience and is deposited with 35 USC. Required under §112 Does not acknowledge that Polynucleotides contained in the deposited material The sequence of the peptide, and also the amino acid sequence of the polypeptide encoded thereby. The columns form part of the present specification and are inconsistent with the description of any of the sequences herein. Whenever we check, we check. Manufacture, use, or sell the deposited material License may be required, and such a license is granted here. Absent.   The present invention further comprises or has the deduced amino acid sequence of FIGS. 1 and 2. Chemokine polypep having amino acid sequence encoded by deposited cDNA And also fragments, analogs and the like of such polypeptides. Related to derivatives.   Encoded by the polypeptide of FIG. 1 and the deposited cDNA. "Fragments", "derivatives" and "analogs" The term "is used to describe substantially the same biological function or activity as such a polypeptide. Means a polypeptide that retains its properties. Therefore, analogs have protein Activating by cutting a portion to produce an active mature polypeptide Protein protein.   The chemokine polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide. Alternatively, it may be a synthetic polypeptide, preferably a recombinant polypeptide.   Encoded by the polypeptide of FIG. 1 and the deposited cDNA. The fragment, derivative or analog of the polypeptide is (i) one or More amino acid residues are homologous or non-homologous amino acid residues (preferably, Amino acid residue), and such a substituted amino acid residue May or may not be coded by (Ii) one or more amino acid residues containing a substituent, or Or (iii) a compound wherein the mature polypeptide increases the half-life of the polypeptide ( Those fused with other compounds, such as, for example, polyethylene glycol), Or (iv) for use in purifying leader or secretory sequences, or mature polypeptides Sa Additional amino acids, such as the sequence or the protein sequence It may be fused to a polypeptide. Such fragments, derivatives and And analogs are deemed to be within the purview of those skilled in the art from the teachings herein.   The polypeptides and polynucleotides of the invention are provided in an isolated form Preferably, it is purified to homogeneity.   The term "isolated" refers to the substance that is in its original environment (e.g., If present, it has been removed from the natural environment). For example, living A naturally occurring polynucleotide or polypeptide in an animal is isolated But not isolated from some or all of the co-existing substances in the natural system The same polynucleotide or polypeptide has been isolated. Such a polynucus The leotide can be part of a vector and / or such a polynucleotide Or polypeptide can be part of the composition, and such vectors or The composition is still isolated in that it is not part of its natural environment.   The present invention also relates to a vector containing the polynucleotide of the present invention, a vector of the present invention. Genetically engineered host cells and recombinant techniques for polypeptides of the invention It also relates to manufacturing by.   The host cell may be, for example, a cloning vector or an expression vector. Genetically engineered (transduced or transduced) with the vectors of the invention Formed or transfected). The vector is, for example, a plasmid. It can be in the form of a sumid, virus particle, phage, and the like. The engineered host cells Activating the promoter, selecting transformants, or Ckβ-4 and And a conventional nutrient medium modified to be suitable for amplifying the Ckβ-10 gene. Can be nourished. Culture conditions such as temperature, pH, etc. are selected for expression. The culture conditions previously used for the selected host cells and will be apparent to those skilled in the art.   The polynucleotides of the present invention can be used to produce peptides by recombinant techniques. can do. Thus, for example, the polynucleotide can be expressed as a polypeptide. The expression vector can be included in any one of a variety of expression vectors. That Such vectors include chromosomal, non-chromosomal and synthetic DNA sequences, eg, SV4 0 derivative; bacterial plasmid; phage DNA; baculovirus; yeast plus Mid; a vector obtained from the combination of a plasmid and phage DNA; Virus DNA such as a, adenovirus, fowlpox virus, pseudorabies Is included. However, any other vector is not capable of replicating in the host. And can be used as long as it is viable.   The appropriate DNA sequence can be inserted into the vector by various methods. General The DNA sequence may be converted to a suitable restriction endonuclease moiety by methods known in the art. Insert in place. Such and other methods are deemed to be within the purview of those skilled in the art. It is.   The DNA sequence of the expression vector can be operated with an appropriate expression control sequence (promoter) To cause mRNA synthesis. As a representative example of such a promoter, These include: LTR or SV40 promoter,E. coli. lacMa Ortrp, Phage lambda P_LPromoters and prokaryotic or eukaryotic cells or Other promoters known to control gene expression in those viruses Tar. Expression vectors also provide a ribosome binding site for translation initiation and transcription termination. Including concessions. The vector may also include appropriate sequences for amplifying expression.   In addition, the expression vector may be a dihydrofolate reductor for eukaryotic cell culture. Such as resistance to tase or neomycin, orE.coliThen Tetra Rhino Transformed host cells, such as culin or ampicillin resistance One or more selectable to provide a phenotypic trait for cell selection It preferably contains a marker gene.   Suitable DNA sequences as described above, and also suitable promoters or Using the vector containing the control sequence to transform a suitable host, It can allow the host to express the protein.   Representative examples of suitable hosts include the following:E.coli,Streptomyc es ,Salmonella typhimuriumBacterial cells such as; fungal cells such as yeast ;DrosophilaandSf9Insect cells such as CHO, COS or Bow Animal cells such as es melanoma; plant cells and the like. Selection of an appropriate host It is believed to be within the purview of those skilled in the art from the teachings in the specification.   In particular, the present invention also includes one or more of the sequences broadly described above. Also included are recombinant constructs consisting of The construct may have the sequence of the invention in a forward or reverse orientation. A vector, such as a plasmid or viral vector, inserted at Comprising. In a preferred aspect of this embodiment, the construct further comprises, for example, It comprises control sequences, including a promoter operably linked to the sequence. suitable Many different vectors and promoters are known to those of skill in the art and are commercially available . The following vectors are given as examples. For bacteria: pQE70, pQE60, pQE- 9 (Qiagen), pBS, pD10, phagescript, psiX17 4. pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH4 6A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR5 40, pRIT5 (Pharmacia). For eukaryotes: pWLNEO, pSV2CAT, pO G44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, pSV L (Pharmacia). However, for any other plasmid or vector, It can be used as long as it is replicable and viable in the host.   The promoter region is a CAT (chloramphenicol transferase) vector. Using any vector with a vector or a selection marker, You can choose from children. Two suitable vectors are PKK232-8 and And PCM7. Individually named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_R, P_LAnd trp. Eukaryotic promoters , CMV immediate early, HSV thymidine kinase, early and later SV40, LTR from retrovirus, and mouse metallothionein- I. Selection of appropriate vectors and promoters is well within the skill of the artisan. Within the range of the bell.   In a further aspect, the present invention relates to a host cell comprising the above-described construct. The inn Primary cells are higher eukaryotic cells such as mammalian cells, lower eukaryotic cells such as yeast cells Or the host cell can be a prokaryotic cell, such as a bacterial cell. Building Transfection of host cells with calcium phosphate transfection, DEAE-de By kisstran-mediated transfection or electroporation be able to. (Davis, L., Dibner, M., Battey, L., Basic Methods in Molecular Biology (1986)).   Encoded by the recombinant sequence using the construct in the host cell in the usual manner Gene products can be produced. Alternatively, the polypeptide of the invention may be It can be produced synthetically by a conventional peptide synthesizer.   Mature proteins can be obtained from mammalian cells, yeast, or bacteria under the control of appropriate promoters. , Or in other cells. Such proteins A cell-free translation system is also required for production using RNA obtained from the light DNA construct. Also can be used. Cloni suitable for use in prokaryotic and eukaryotic hosts And expression vectors are described in Sambrook et al., Molecular Cloning: A Laborato. ry Manual, Second Edition, Cold Spring Harbor, New York, (1989). And this disclosure forms a part of the present specification.   Transcription of a DNA encoding a polypeptide of the present invention by higher eukaryotes may be It is increased by inserting a Hanser sequence into the vector. Enhancers are professional DNA, usually about 10 to 300 bp, that acts on the motor to increase its transcription Is a cis-acting element. Examples include the late side of the replication origin of bp 100-270. SV40 enhancer, cytomegalovirus early promoter enhancer Sir, polyoma enhancer on the late side of the replication origin, and adenovirus Suenhansa included.   Typically, recombinant expression vectors contain an origin of replication, and a host cell Selectable markers that allow for animation, such asE.coliThe Ampicily Resistance gene andS. cerevisiae Inversion of TRP1 gene and downstream structural sequence Includes promoters derived from highly expressed genes to drive transcription Will be. Such promoters are, inter alia, 3-phosphoglycerate Glycolytic enzymes such as PGK, α-factor, acid phosphatase, It can be obtained from an operon that encodes a protein. Heterologous structure The construction sequence may include translation initiation and termination sequences, and preferably Fine With a leader sequence capable of directing secretion into the periplasmic space or extracellular medium , Built in the appropriate phase. Optionally, the heterologous arrangement is desired Properties, such as stabilization of the expressed recombinant product or simplified purification A fusion protein containing an N-terminal identification peptide can be encoded.   Expression vectors useful for use in bacteria include those encoding the desired protein. The synthetic DNA sequence, along with the appropriate translation initiation and termination signals, is combined with a functional promoter. Constructed by insertion into an operable reading phase having a That The vector may be used to ensure the maintenance of the vector and, if desired, the host. One or more phenotypic selectable markers to confer amplification within And a replication origin. Source suitable for transformation Nuclear hosts include:E.coli,Bacillus subtilis,Salmonella typhimurium, And Various species within the genus Pseudomonas, Streptomyces, and Staphylococcus Although species are included, others can also be used as selection agents.   As a representative, but non-limiting example, vectors useful for use in bacteria are Genetic elements of the well-known cloning vector pBR322 (ATCC 37017) A selectable marker and a bacterial strain obtained from a commercially available plasmid comprising It may comprise a replication origin. Such commercially available vectors include, for example, pKK 223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pG EM1 (Promega Biotec, Madison, Wis., USA). These pBR Combining a 322 "bone nucleus" portion with a suitable promoter and the structural sequence to be expressed Let me know.   Transform an appropriate host strain and set the host strain at the appropriate cell density. After growth, the selected promoter is replaced by an appropriate method (e.g., temperature shift). Or chemical induction) and the cells are cultured for an additional period.   Cells are generally harvested by centrifugation and by physical or chemical methods Break down and retain the resulting crude extract for further purification.   Microbial cells used for protein expression may be subjected to freeze-thaw cycles, sonication Destroyed by any conventional method, including mechanical disruption or use of cell lysing agents so Such methods are well known to those skilled in the art.   Various mammalian cell culture systems are also used to express recombinant proteins can do. Examples of mammalian expression systems include Gluzman, Cell, 23: 175. (1981), COS-7 line of monkey kidney fibroblasts, And other cell lines capable of expressing compatible vectors, such as C12 7, 3T3, CHO, HeLa and BHK cell lines are included. Mammalian expression vectors The promoter is the origin of replication, the appropriate promoter and enhancer, and any Necessary ribosome binding sites, polyadenylation sites, splice donors and It should also not contain sceptor sites, transcription termination sequences, and non-transcribed sequences 5 'adjacent. Will be. DNA sequence obtained from SV40 splicing, Denenylation sites can be used to provide the required non-transcribed genetic elements.   Chemokine polypeptide is precipitated by ammonium sulfate or ethanol, acid extraction , Anion or cation exchange chromatography, phosphocellulose chromatography Chromatography, hydrophobic interaction chromatography, affinity chromatography Fee, hydroxylapatite chromatography, and lectin chromatography Can be recovered and purified from recombinant cell culture by a method that includes chromatography. Can be. If necessary, perform the protein regeneration step Can be used to complete the installation. Finally, high performance liquid chromatography (HPLC) can be used for the final purification step.   The chemokine polypeptide of the present invention may be a naturally purified product or a chemically synthesized product. It can be the product of a synthetic process or from a prokaryotic or eukaryotic host (e.g., in culture). (By bacteria, yeast, higher plants, insects and mammalian cells) Can be manufactured. The polypeptide of the present invention depends on the host used in the recombinant production method. Peptides can be glycosylated or not glucosylated. Of the present invention The polypeptide may also include an initial methionine amino acid residue.   Chemokine polypeptides are adjuncts during cancer chemotherapy and against leukemia. As a protective treatment, it can be used to inhibit colony formation of bone marrow stem cells. Wear.   The chemokine polypeptide is also characterized by keratinocyte hyperproliferation. Used to inhibit epidermal keratinocyte proliferation as a treatment for psoriasis Can also.   The chemokine polypeptide can also be a host defense cell, eg, a cytotoxic T cell. And treat solid tumors by stimulating macrophage invasion and activation. It can also be used to place. They are also resistant to chronic infections, for example, Accommodation against mycobacterial infection by attracting and activating mycobacterial leukocytes Can also be used to increase primary defense.   The chemokine polypeptide may also inhibit proliferation of T cells by inhibiting biosynthesis of IL2. Can be used to treat autoimmune diseases and lymphocytic leukemia by inhibiting Can also be used.   Ckβ-4 and Ckβ-10 also clear debris, Medium due to the recruitment of inflammatory cells that promotes lost connective tissue and excess TGFβ. It can also be used in wound healing by controlling mediated fibrosis. This In the same way, Ckβ-4 and Ckβ-10 are also used for cirrhosis, osteoarthritis and Can also be used to treat other fibrotic disorders, including and fibrosis You. The chemokine polypeptides are also used in schistosomiasis, trichinosis and ascariasis. Have a distinctive function of killing parasite larvae that invade tissues. It also increases the presence of acid spheres. They are also responsible for the activation and distribution of various hematopoietic progenitor cells. By regulating hematopoiesis, it can also be used to regulate hematopoiesis.   Chemokine polypeptides may also be used as inhibitors of angiogenesis They can also have an antitumor effect.   The chemokine polypeptides of the present invention also provide for other components with similar biological activity. It is also useful for identifying offspring. Examples of screening in this regard are known By synthesizing oligonucleotide probes using the DNA sequence of Isolating the coding region of a gene. Sequence complementary to the sequence of the gene of the present invention Labeled oligonucleotide having human cDNA, genomic DNA or mR Which of the libraries to use to screen the library of NA Determine if the probe will hybridize to the member.   The invention also relates to altered levels of the polypeptide, or such polypeptides. To quantitatively and qualitatively detect mRNAs that provide It also relates to diagnostic assays. Such assays are well known in the art, and Includes LISA assays, radioimmunoassays and RT-PCR. Said a The levels of polypeptides, or their mRNAs, detected in the assay Elucidation of the significance of polypeptides in various diseases, and altered levels of polypeptides Peptides can be used in the diagnosis of diseases where they can be important.   The present invention provides a method for identifying a receptor for a polypeptide. The receptor The gene to be loaded can be identified by expression cloning. Polyadeni Prepared from cells that respond to the polypeptide, and Dividing the cDNA library to be pooled into COS cells or the polypeptide Used to transfect other cells that do not respond to Glass slide Exposing the transfected cells grown above to a labeled polypeptide . The polypeptide may be iodinated or recognized for site-specific protein kinases. Labeling can be performed by various methods, including inclusion of a recognition site. Fixed and a Following incubation, the slides are subjected to autoradiographic analysis. Identify positive pools, prepare subpools, and repeat subpooling and respooling. Using the cleaning method, transfect again and ultimately estimate A single clone is obtained which encodes a receptor for the receptor. With other methods to identify the receptor To allow the labeled polypeptide to bind to the cell membrane with photoaffinity or to accept Preparations expressing body molecules can be extracted. Cross-linked substances Separate by PAGE analysis and expose to X-ray film. Receptor for the polypeptide The labeled labeling complex is excised, split into peptide fragments, and Can be subjected to sequencing. Use the amino acid sequence obtained from microsequencing. To design a set of oligonucleotide probes and construct a cDNA library Screening will identify genes encoding putative receptors U.   The present invention enhances the interaction of polypeptides with their identified receptors. To identify drugs that block (agonists) or block (antagonists) And a method of screening for a drug. An agonist is the polypeptide Compounds that increase natural biological function, but antagonists Eliminate unnecessary functions. By way of example, mammalian cells expressing the receptor for the polypeptide may be used. A vesicle or membrane preparation is prepared in the presence of a drug in the presence of a labeled chemokine polypeptide, e.g., Will be incubated with radioactivity. Then enhance this interaction, Will be able to measure the ability of the drug to block.   Potential antagonists include antibodies, and in some cases, polypeptides. Oligonucleotides that bind to the peptide. Different potential antagonists Examples are negative dominant mutants of the polypeptide. Negative dominant sudden change Variants bind to the receptor for the wild-type polypeptide, but retain biological activity Not a polypeptide.   Assays for detecting negative dominant mutants of a polypeptide include Multiwell chemotaxis chamber with rupyrrolidone-free polycarbonate membrane Using the bar, potential antagonists / inhibitors or agonists In the presence and absence of molecules, the ability of a polypeptide to chemoattract leukocytes An in vitro chemotaxis assay to measure is included.   Antisense constructs made using antisense technology are also potential Certain antagonists. Using antisense technology, triple helix formation or Can regulate gene expression by antisense DNA or RNA , Both methods rely on the binding of a polynucleotide to DNA or RNA. Good. For example, a polynucleotide sequence encoding a mature polypeptide of the invention. Using the 5 'coding portion, an antisense RNA oligonucleotide of about 10-40 base pairs in length is used. Design gonucleotides. DNA oligonucleotides are Designed to be complementary to the child region (triple helix-Lee et al., Nucl. s., 6: 3073 (1979); Cooney et al., Science, 241: 456 (19). 88); and Dervan et al., Science, 251: 1360 (1991)). This prevents transcription and production of the polypeptide. Antisense RN A-ori The oligonucleotides hybridize to the mRNA in vivo and produce mRNA components. Blocking translation of the offspring into the polypeptide (antisense-Okano, J. Neuroch em., 56: 560 (1991); Oligodeoxynucleotides as Antisense Inh. ibitors of Gene Expression, CRC Press, Boca Raton, FL (198 8)). The above oligonucleotides are also delivered to cells, which RNA or DNA is expressed in vivo to produce a polypeptide. Can be inhibited.   Another potential antagonist has lost its biological function, , Nevertheless, recognizes and binds to the receptor for the polypeptide, thereby , Naturally or synthetically, of a polypeptide that effectively blocks its receptor Decorated analogs, peptide derivatives of polypeptides. Peptide induction Examples of bodies include, but are not limited to, small peptides or peptides. De-like molecules are included.   The antagonists are useful in autoimmune diseases and chronic inflammatory diseases and infections. Macrophages and their precursors, as well as neutrophils, basophils, Lymphocytes and some T cell subsets, such as activation and CD8 cell damage Used to inhibit the chemotaxis and activation of sexual T cells and natural killer cells can do. Examples of autoimmune diseases include rheumatoid arthritis, multiple sclerosis, And insulin-dependent diabetes. Some infections include mononuclear phagocyte Silicosis, sarcoidosis, idiopathic pulmonary fibrils due to impairment of recruitment and activation Disease, idiopathic hyper-eosin-favorable syndrome by preventing eosinophil production and migration, Preventing migration of clophage and production of chemokine polypeptides of the invention Endotoxin shock. The antagonist may also be Used to treat atherosclerosis by preventing monocyte infiltration You can also.   The antagonists also include mast cells and halophilics induced by chemokines. Inhibiting degranulation of base spheres and release of histamine It can also be used to treat allergic reactions that are mediated.   The antagonists may also inhibit inflammation by preventing monocytes from attracting to the wound area. Can also be used to treat They also have acute and chronic inflammation Normal pulmonary disease is associated with mononuclear phagocytic bone formation in the lung, Can also be used to regulate the number of macrophages.   Antagonists may also prevent the attraction of monocytes into synovial fluid in patient joints. And can also be used to treat rheumatoid arthritis. Monocyte flow Entry and activation play important roles in the pathogenesis of both degenerative and inflammatory arthritis Carry.   The antagonists may also interfere with the biosynthesis of other inflammatory cytokines, primarily Used to prevent deleterious cascades caused by IL-1 and TNF You can also. In this way, the antagonist is also used to prevent inflammation. Can also be used. The antagonist is also induced by a chemokine, It can also be used to block fever independent of prostaglandins.   The antagonists are also useful in diseases of bone marrow failure, such as aplastic anemia and spinal cord. It can also be used to treat myelodysplastic syndrome.   The antagonists also prevent eosinophil accumulation in the lungs, thereby reducing asthma and And can also be used to treat allergies. The antagonist is Also, a composition with a pharmaceutically acceptable carrier, for example, a carrier as described below. It can also be used in objects.   Chemokine polypeptides and agonists or antagonists of the invention It can be used in combination with a suitable pharmaceutical carrier. Such a composition is A therapeutically effective amount of the polypeptide, and a pharmaceutically acceptable carrier or excipient. Comprising. Such carriers include, but are not limited to, saline Water, buffered saline, dextrose, water, glycerol, ethanol, and And combinations thereof. The formulation should suit the mode of administration.   The present invention also relates to one or more of the components of the pharmaceutical composition of the present invention, Pharmaceutical packs or kits comprising one or more containers are also provided. So Manufacture, use or sale of pharmaceutical or biological products in connection with containers such as Notifications may be given in the form prescribed by the regulatory authorities, which may Represents the approval of the appropriate department for manufacture, use or sale for administration to a healthcare professional. further The polypeptides of the present invention can be used with other therapeutic compounds.   The pharmaceutical composition may be topical, intravenous, intraperitoneal, intramuscular, intratumoral, subcutaneous, intranasal or Can be administered in any convenient way, such as by the intradermal route. The polypeptide The drug is administered in an amount effective to treat and / or prevent the particular indication. Generally, the polypeptide is administered in an amount of at least about 10 μg / kg (body weight), Most often they are administered in an amount not to exceed about 8 mg / kg (body weight) per day. Will be. In most cases, the dosage should be About 10 μg / kg to about 1 mg / kg (body weight) daily.   The chemokine polypeptide and agonist or antagonist are In vivo development of such polypeptides, often referred to as "child therapy" At present, it can be used according to the invention.   Thus, for example, a patient-derived cell can be used to encode a polypeptide ex vivo. After the manipulation with the polynucleotide (DNA or RNA) to be Give to the patient to be treated with the peptide. Such methods are well-known in the art. For example, a retroviral particle containing RNA encoding the polypeptide of the present invention. Through use, cells can be manipulated by methods known in the art.   Similarly, polypeptides can be used in vivo, for example, by methods known in the art. The cells can be manipulated in vivo for different expression. Known in the industry As has been described, cells are treated in vivo to convert the polypeptide in vivo. Containing RNA encoding the polypeptide of the present invention for expression in Producer cells for producing rovirus particles can be administered to a patient. That These and other methods for administering the polypeptides of the invention by such methods and others Will be apparent to those skilled in the art from the teachings of the present invention. For example, manipulate cells Expression vehicles for use include those other than retroviruses, for example, a suitable delivery vehicle. Once combined with the cells, they can be used to manipulate cells in vivo. Adenovirus that can be used.   The sequences of the present invention are also valuable for chromosome identification. Individual sequencing of the sequence Specifically targeted to specific locations on the body and allowed to hybridize. Wear. Moreover, there is currently a need to identify specific sites on the chromosome. Actual distribution Chromosome marking reagents based on column data (repeated polymorphisms) currently Hardly available to mark. The mapping of DNA to chromosomes according to the invention Ping is an important first step in relating those sequences to genes associated with the disease. is there.   Briefly, PCR primers derived from cDNA (preferably 15-25 bp) Can be mapped to a chromosome. cDNA One or more exons in genomic DNA are spanned (s) using computer analysis. Quick selection of primers that do not pan) complicates the amplification process . These primers are then transferred to somatic cell hybrids containing individual human chromosomes. Used for PCR screening. Including the human gene corresponding to the primer, Only those hybrids will give the amplified fragment.   PCR mapping of somatic cell hybrids returns specific DNA to specific chromosomes. A quick way to belong. Using the same oligonucleotide primer Utilizing the present invention, sublocalization can be performed on specific chromosomes or large Achieved in a similar manner using a panel of fragments from a pool of genomic clones can do. Others that can be similarly used to map to that chromosome Methods include in situ hybridization and labeled flow-sourcing. Pre-screening using chromosomes (flow-sorted) Hybridization to construct a heterologous cDNA library Includes selection.   Fluorescence of cDNA clones to metaphase chromosomal spreads in situ hybridization Chromosome location (FISH) can provide accurate chromosome locations in one step You. This technique can be used with cDNAs as short as 500 or 600 bases. Yes, but clones larger than 2,000 bp are sufficient for simple detection. It is likely to bind to a unique chromosomal location with null strength. FISH is ES Requires the use of clones that yield T, and the longer the better. For example, 2,000 bp is better, 4,000 is better, and more than 4,000 Probably not necessary to get good results at a reasonable time rate. To review this technique Verma et al., Human Chromosomes: a Manual of Basic Techniques, Perga. mon Press, New York (1988).   Once a sequence has been mapped to a precise chromosomal location, the physical Locations can be associated with genetic map data. Such data, for example, For example, V. McKusick, Mendelian Inheritance in Man (Johns Hopkins Univ (available online via ersity Welch Medical Library) It is. Next, the relationship between a gene mapped to the same chromosomal region and a disease is determined. Confirmed by binding analysis (coinheritance of physically adjacent genes) .   Next, the cDNA or genotype between affected and unaffected individuals is determined. It is necessary to determine the differences in the sequence. The mutation is in some or all of the affected individuals If not found in any normal individual, Mutations appear to cause disease.   Currently, analysis of physical and genetic mapping suggests that CDNAs localized correctly in chromosomal regions represent 50-500 possible causative sources. It could be one of the genes. (This is a 1-megabase mapping analysis and And one gene per 20 kb).   Polypeptides, their fragments or other derivatives, or their Use analogs, or cells expressing them, as immunogens to Antibodies can be produced. These antibodies are, for example, polyclonal or Can be a monoclonal antibody. The invention also includes chimeric, single chain, and human Of Activated Antibodies, and also Fab Fragments, or Fab Expression Libraries Things are also included. Various methods known in the art may be used to identify such antibodies and fragments. Can be used in the manufacture of   Antibodies raised against polypeptides corresponding to the sequences of the present invention are polypeptides The polypeptide directly into the animal, or the polypeptide into the animal, preferably a human. It can be obtained by administering to non-animal animals. Then, like that The resulting antibody will bind to the polypeptide itself. In this way, Even sequences that encode only fragments of the polypeptides can be completely native polypeptides. It can be used to produce antibodies that bind tide. Then such Using antibodies to isolate a polypeptide from a tissue that expresses the polypeptide be able to.   Monoclonal antibodies are prepared using anti-cells produced by continuous cell line culture. All body giving techniques can be used. Examples include hybridoma technology (Ko hler and Milstein, 1975, Nature, 256: 495-497), trio Trioma technology, human B cell hybridoma technology (Kozbor et al., 1983, Immm unology Today 4:72), and E for producing human monoclonal antibodies. BV-Hybridoma technology (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). .   Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) Is suitable for producing single chain antibodies against the immunogenic polypeptide products of the invention. I can do it.   The present invention will be further described with reference to the following examples; It should be understood that the present invention is not limited to such an embodiment. All parts or quantities, especially Unless otherwise indicated, units are by weight.   To facilitate understanding of the following examples, some frequently occurring methods and And / or describe terms.   “Plasmids” are preceded by a lower case p and / or And / or indicated by numbers. The starting plasmids herein are commercially available, limited Publicly available on an unspecified basis or obtained by published methods Can be constructed from possible plasmids. Plus, equivalent to the described plasmid Mids are known in the art and will be apparent to those skilled in the art.   “Digestion” of DNA involves touching it with a restriction enzyme that acts only on certain sequences in the DNA. Indicates that the medium is cut. Various restriction enzymes used herein are commercially available. , Their reaction conditions, cofactors and other requirements are known to those skilled in the art. Used for For analytical purposes, typically 1 μg of plasmid in about 20 μl of buffer solution is used. Or a DNA fragment is used with about 2 units of enzyme. Plasmid construction For the purpose of isolating DNA fragments for 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme. For certain restriction enzymes Appropriate buffers and substrate weights are specified by the manufacturer. About 1 hour at 37 ° C Incubation times are usually used, but may vary according to the supplier's instructions. Can be. After digestion, the reaction is directly electrophoresed on a polyacrylamide gel. Isolate the desired fragment.   Size separation of the cleaved fragments is described by Goeddel, D et al., Nucleic Acids Re. s., 8: 4057 (1980). Perform using a gel.   An “oligonucleotide” is a single-stranded polydeoxy that can be chemically synthesized. Shows a xynucleotide, or two complementary polynucleotide strands. like that Since synthetic oligonucleotides do not have a 5 'phosphate, the presence of kinases In the presence, without adding a phosphate with ATP, to another oligonucleotide Will not ligate. Synthetic oligonucleotides are not dephosphorylated Will ligate to the new fragment.   "Ligation" refers to the phosphodiester between two double-stranded nucleic acid fragments. (Maniatis, T. et al., Ibid., P. 146). Especially saying Unless otherwise described, ligation should be performed on the DNA fragment to be ligated. With 10 units of T4 DNA ligase ("ligase") per 0.5 μg of equimolar amount Can be accomplished using known buffers and conditions.   Unless otherwise stated, transformations were performed by Graham, F. and Va. n der Eb, A., Virology, 52: 456-457 (1973). I went as it was.                                 Example 1                       Bacterial expression and purification of Ckβ-4   First, the DNA sequence encoding Ckβ-4, ATCC No. 75848, was 5 ′ and 3 ′ sequences of the processed Ckβ-4 protein (presumed signal (Without peptide sequence) Amplify. Additional nucleotides corresponding to Ckβ-4 were replaced with 5 ′ and 3 ′ sequences, respectively. Was added to The 5 'oligonucleotide primer has the sequence: And encodes the SphI restriction enzyme site (printed in bold), followed by the mature protein Ckβ-4 coding sequence of 17 nucleotides starting from the second nucleotide of the sequence (Underlined type). The ATG codon is included in the SphI site. ATG In the next codon that follows, the first base is from the SphI site and the remaining two salts The groups are located at the second and third bases of the first codon of the putative mature protein. Corresponding. As a result, the first base in this codon is compared to the original sequence. As a result, G changes to C, and consequently E is replaced with Q in the recombinant protein. Be replaced. 3 'sequence: Is the sequence complementary to the BamH1 site (bold print) followed by the stop codon Contains a 21 nucleotide gene-specific sequence. The restriction enzyme site is The current vector pQE-70 (Qiagen, Inc. 9259 Eton Ave., Chatsworth, CA 91311). pQE-70 is resistant to antibiotics ( Amp^r), Bacterial origin of replication (ori), IPTG-regulatable promoter operation (P / O), ribosome binding site (RBS), 6-His tag and restriction enzyme site Code. Then, pQE-70 was digested with SphI and BamH1. amplification The ligated sequence is ligated to pQE-70, and the histidine tag and RBS are With the sequence to be loaded. FIG. 8 is a schematic diagram of this arrangement. Shows a typical display. The ligation mixture was then used in Sambrook, J. Et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laborat. ory Press (1989) by the trademark M15 from Qiagen. Available at / rep4E. coli.The strain was transformed. M15 / rep4 is plastic Contains multiple copies of the sumid pREP4, which expresses the lacI repressor, In addition, kanamycin resistance (Kanmycin^r) Also give. Transformants, if they are L Ampicillin / kanamycin resistant colonies, identified by their ability to grow on B plates I chose Ronnie. Plasmid DNA was isolated and confirmed by restriction analysis. Place Colonies containing the desired construct were selected for both Amp (100 ug / ml) and Kan (25 ug / ml). Growing overnight (O / N) in liquid culture in supplemented LB medium. The O / N culture In use, large cultures were seeded at a ratio of 1: 100 to 1: 250. Cells Optical density 600 of 0.4 to 0.6 (OD.⁶⁰⁰). Then, IPTG (" Isopropyl-BD-thiogalactopyranoside ") at a final concentration of 1 mM. Added. IPTG inactivates the P / I by inactivating the lacI repressor. Induces O clearing and increases gene expression. Cells for an additional 3-4 hours Propagated. The cells were then collected by centrifugation. Cell pellet Solubilized in 6 moles of guanidine HCl, a pick agent. After clarification, Et al., Conditions that allow tight binding by proteins containing a 6-histidine tag. Ckβ solubilized by chromatography on a nickel-chelate column -4 was purified. (Hochuli, E. et al., J. Chromatography 411: 177-18. 4 (1984)). Ckβ-4 (purity> 6 mol guanidine HCl (pH 5.0) 98%) eluted from the column. Regeneration of protein from GnHCl has several consequences. Protoco Can be achieved by (Jaenicke, R. and Rudolph, R., Prote in Structure-A Practical Approach, IRL Press, New York (1 990)). First, GnHCL is removed using step dialysis. Or The purified protein isolated from the Ni-chelate column was converted to reduced linear Gn. It can be bound to a second column through an HCL gradient. The protein After regeneration while binding the material to the column, 250 mM imidazole, 150 mM NaCl, 25 mM Tris-HCl (pH 7.5) and 10% glycerol. Elute with the buffer solution containing. Finally, the soluble protein was replaced with 5 mM ammonium bicarbonate. Dialysis against a storage buffer containing urea.                                 Example 2                     Bacterial expression and purification of Ckβ-10   First, the DNA sequence encoding Ckβ-10, ATCC No. 75849, 5 ′ and 5 ′ of the processed Ckβ-10 protein (no signal peptide sequence) And 3 ′ sequence, and a PC corresponding to the 3 ′ vector sequence of the Ckβ-10 gene Amplify using the R oligonucleotide primer. Corresponding to Ckβ-10 Additional nucleotides were added to the 5 'and 3' sequences, respectively. 5 'oligonucleo The tide primer has the sequence: And encodes the SphI restriction enzyme site (printed in bold), followed by the mature protein 19 nucleotide Ckβ-10 coding sequence starting from the sequence )including. The ATG codon is included in the SphI site. 3 'sequence: Is the sequence complementary to the BamH1 site (bold print) followed by the stop codon Contains a gene-specific sequence of 19 nucleotides. The restriction enzyme site is The current vector pQE-70 (Qiagen, Inc. 9259 Eton Ave., Chatsworth, CA 91311). pQE-70 is resistant to antibiotics ( Amp^r), Bacterial origin of replication (ori), IPTG-regulatable promoter operation Ta -(P / O), ribosome binding site (RBS), 6-His tag and restriction enzyme site Code. Then, pQE-70 was digested with SphI and BamH1. Amplified The ligated sequence is ligated to pQE-70 to encode a histidine tag and RBS. With the sequence to be inserted. FIG. 10 shows a schematic representation of this sequence. . The ligation mixture is then used to prepare the protein in Sambrook, J. et al., Molecula r Cloning: A Laboratory Manual, Cold Springing Laboratory Press (1 989) by the procedure described in Qiagen under the trademark M15 / rep4 PossibleE. coli.The strain was transformed. M15 / rep4 is plasmid pREP 4 multiple copies, which express the lacI repressor and Thin resistance (Kan^r) Also give. Transformants, they are LB plates Select for ampicillin / kanamycin resistant colonies, identified by their ability to grow did. Plasmid DNA was isolated and confirmed by restriction analysis. The desired construct Included colonies were grown in LB medium supplemented with both Amp (100 ug / ml) and Kan (25 ug / ml) Grow overnight in liquid culture in the ground (O / N). Using the O / N culture, The fresh culture was inoculated at a ratio of 1: 100 to 1: 250. Cells are 0.4-0.6 Optical density of 600 (OD)⁶⁰⁰). Then IPTG ("isopropyl -BD-thiogalactopyranoside ") was added to a final concentration of 1 mM. IPTG clears P / O by inactivating the lacI repressor. And induces increased gene expression. Cells were grown for an additional 3-4 hours. The cells were then collected by centrifugation. The cell pellet is treated with a chaotropic agent. Guanidine HCl. After clarification, 6-His Under conditions that allow for strong binding by proteins containing a thidine tag, nickel- Purified solubilized Ckβ-10 by chromatography on a chelating column did. (Hochuli, E. et al., J. Chromatography 411: 177-184 (198 4)). Ckβ-10 (purity> 98%) in 6 moles of guanidine HCl (pH 5.0) Was eluted from the column. Regeneration of protein from GnHCl has several protocols. Can be achieved by (Jaenicke, R. and Rudolph, R., Prote in Structure-A Practical Approach, IRL Press, New York (1 99 0)). First, GnHCL is removed using step dialysis. Or also , Purified protein isolated from a Ni-chelate column was converted to reduced linear GnHC It can be attached to a second column through an L gradient. That protein After regeneration while binding to the column, 250 mM imidazole, 150 mM N aCl, containing 25 mM Tris-HCl (pH 7.5) and 10% glycerol Elute with buffer. Finally, the soluble protein was replaced with 5 mM ammonium bicarbonate. Dialyze against storage buffer containing Next, the protein was converted to SDS-PAG. Analyzed on E-gel.                                 Example 3                 Expression of recombinant Ckβ-4 in COS cells   The expression of the plasmid, Ckβ-4 HA, is based on 1) the SV40 replication origin, Pyricin resistance gene, 3) E. coli origin of replication, 4) Polylinker region, SV4 0 containing the CMV promoter followed by an intron and a polyadenylation site. Obtained from the vector pcDNAI / Amp (Invitrogen). Complete Ckβ-4 precursor And a DNA fragment encoding an HA tag fused in frame to its 3 'end. Cloned into the polylinker region of the vector, Protein expression is directed under the CMV promoter. The HA tag is Corresponding to an epitope derived from the influenza hemagglutinin protein (I. Wilson, H. Niman, R. Heighten, A. Cherenson, M. Connolly, and And R. Lerner, 1984, Cell 37, 767). HA tag to target protein The fusion allows the recombinant protein to be treated with an antibody that recognizes the HA epitope. It is possible to easily detect.   The plasmid construction method is described below:   DNA sequence encoding Ckβ-4, ATCC Issue with two Constructed by PCR in the original EST, cloned using the immer: 5 'Primer Is a HindIII site followed by a 20 nucleotide Ckβ-starting at the start codon. 4 coding sequences; 3 'sequence Is the sequence complementary to the XbaI site, the translation stop codon, the HA tag, and Ckβ -Contains the last 20 nucleotides of the coding sequence (not including the stop codon) . Thus, the PCR product has a HindIII site, a Ckβ-4 coding sequence, followed by an in-frame Tag, translation termination stop codon next to the HA tag, and an XbaI site Including rank. DNA fragment and vector amplified by PCR, pcDN AI / Amp was digested with HindIII and XbaI restriction enzymes and ligated. The ligation mixture was transferred to E. coli strain SURE (Stratagene Cloning System). ms, 11099 North Torrey Pines Road, La Jolla, CA 92037 (Available from Co., Ltd.) and the transformed culture Articles were placed on ampicillin medium plates to select for resistant colonies. Plasmi DNA is isolated from transformants and analyzed for the presence of the correct fragment. And tested by restriction analysis. For expression of recombinant Ckβ-4, DEAE-DEX was used. COS cells were transfected with the expression vector by the TRAN method (JS ambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). Ckβ-4 HA Protein expression was detected by radiolabeling and immunoprecipitation. (E. Harlo w, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Laborat ory Press, (1988)). Two days after transfection, remove cells³⁵S- Labeled with cysteine for 8 hours. The cell culture medium is then collected and the cells are detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% The cells were lysed with NP-40, 0.5% DOC, 50 mM Tris, pH 7.5). (Wil Son, I. et al., Id. 37: 767 (1984)). Cell lysate and culture medium are both Both were precipitated with a monoclonal antibody specific for HA. The precipitated protein Analyzed by SDS-PAGE.                                 Example 4                 Expression of recombinant Ckβ-10 in COS cells   Expression of the plasmid, Ckβ-10 HA, is based on 1) the SV40 replication origin, 3) E. coli replication origin, 4) polylinker region, SV Including a CMV promoter followed by 40 introns and a polyadenylation site; Obtained from the vector pcDNAI / Amp (Invitrogen). Before complete Ckβ-10 A DNA fragment encoding a precursor and an HA tag fused in frame to its 3 'end Fragment was cloned into the polylinker region of the vector, Protein expression is directed under the CMV promoter. The HA tag is For epitopes derived from influenza hemagglutinin proteins such as (I. Wilson, H. Niman, R. Heighten, A. Cherenson, M. Connolly, And R. Lerner, 1984, Cell 37, 767). Protein targeting HA tag Fusion of the recombinant protein with an antigen that recognizes the HA epitope. It can be easily detected by the body.   The plasmid construction method is described below:   The DNA sequence encoding Ckβ-10, ATCC 75849, was Constructed by PCR on the original EST, cloned using Reimer: 5 'primer Is a HindIII site followed by a 19 nucleotide Ckβ-starting at the start codon. Including 10 coding sequences; 3 'sequence Is the sequence complementary to the XbaI site, the translation stop codon, the HA tag, and Ckβ -10 contains the last 19 nucleotides of the coding sequence (not including the stop codon) No. Thus, the PCR product contains a HindIII site, the Ckβ-10 coding sequence, followed by HA tag fused in frame, translation termination stop codon next to the HA tag, and Xba I site. DNA fragment and vector amplified by PCR, pc DNAI / Amp was digested with HindIII and BamH1 restriction enzymes and ligated. I let you. The ligation mixture was transferred to E. coli strain SURE (Stratagene Cloning). Systems, 11099 North Torrey Pines Road, La Jolla, CA 92 037), and the transformed The resulting culture was placed on ampicillin medium plates to select for resistant colonies. Step Rasmid DNA was isolated from transformants and the correct fragment The presence was tested by restriction analysis. For expression of recombinant Ckβ-10, DEA Transfect COS cells with expression vector by E-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: AL) aboratory Manual, Cold Spring Laboratory Press, (1989)). Ck β-10 HA protein expression was detected by radiolabeling and immunoprecipitation. Was. (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold S pring Laboratory Press, (1988)). 2 days after transfection , Cells³⁵Labeled with S-cysteine for 8 hours. The cell culture medium is then collected and Cells were washed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1%  SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5)) Lysed. (Wilson, I., et al., Supra at 37: 767 (1984)). Cell lysate and Both culture media were sedimented with a monoclonal antibody specific for HA. Settling The analyzed proteins were analyzed by SDS-PAGE.                                 Example 5                   Using the baculovirus expression system                     Cloning and expression of Ckβ-10   DNA sequence encoding full length Ckβ-10 protein, ATCC VII No. 5849, PCR oligonucleotides corresponding to the 5 ′ and 3 ′ sequences of the gene Amplification was performed using primers.   The 5 'primer has the sequence: And a BamHI restriction enzyme site (printed in bold), followed by translation in eukaryotic cells. Contains 12 nucleotides similar to the signal available to initiate translation (J. Mol. Biol. 1987,196947-950, Kozak, M.) and immediately behind Ckβ-1. 0 is the first 6 nucleotides of the coding sequence (the translation initiation codon "ATG" is underlined) pull).   The 3 'primer has the sequence: And a cleavage site for the restriction endonuclease Asp781, and Ckβ-1 It contains 19 nucleotides complementary to the 3 'untranslated sequence of the 0 gene. Amplified The sequence was compared to a commercially available kit ("Geneclean", BIO 101 Inc., La Jolla, Was isolated from a 1% agarose gel using Ca.). Then, the fragment After digestion with endonucleases BamHI and Asp781, 1% agaro It was then purified on a gel. This fragment is named F2.   The vector pRG1 (a modification of the pVL941 vector, discussed below) was transformed into a baculo Used for expression of Ckβ-10 protein using a viral expression system (Revi Reviews include Summers, MD and Smith, GE. 1987, A manual of meth ods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555). this The expression vector is Autographa California Nuclear Polyhedrosis Russ group (AcMNPV) strong polyhedrin promoter followed by control Includes recognition sites for the restriction endonucleases BamHI and Asp781. Simian Use the polyadenylation site of virus (SV) 40 for efficient polyadenylation . For easy selection of recombinant viruses, β-galactosidase from E. coli was used. The gene is a polyhedrin gene followed by a polyadenylation signal of the polyhedrin gene. Insert in the same direction as the motor. Cotransfected Viral sequence for cell-mediated homologous recombination of wild-type viral DNA The columns are adjacent on both sides of the polyhedral array. Many other baculovirus vectors, Instead of pRG1 such as pAc373, pVL941, and pAcIM1 Could be used (Luckow, VA and Summers, MD, Virol ogy, 170: 31-39).   After digesting the plasmid with the restriction enzymes BamHI and Asp781, it is known in the art. Was dephosphorylated using calf intestinal phosphatase. Then, DN A was isolated from a 1% agarose gel. This vector DNA is named V2. .   Fragment F2 and dephosphorylated plasmid V2 were converted to T4 DNA ligase Was lit. Next, E. coli HB101 cells were transformed. , A bacterium containing a plasmid having the Ckβ-10 gene (pBacCkβ-10) Identification was performed using elementary BamHI and Asp781. Cloned fragment Was confirmed by DNA sequencing.   Using the lipofection method, 5 μg of plasmid pBacCkβ-10 was commercially available. Linearized baculovirus ("BaculoGold")^TMBaculovirus DNA ", Pha rmingen, San Diego, Calif.) with 1.0 μg (Felgn er et al., Proc. Natl. Acad. Sci. ScL USA, 84: 7413-7417 (1987). )).   BaculoGold^TM1 μg of viral DNA and plasmid pBacCkβ-105 μg of serum-free Grace's medium (Life Technologies Inc., Gaithers) burg, MD) mixed in sterile wells of a microtiter plate containing 50 μl . Then, 10 μl of Lipofectin and 90 μl of Grace's medium were added. , Mixed and incubated for 15 minutes at room temperature. Then, transfection Seeded in a 35 mm tissue culture plate containing 1 ml of serum-free Grace's medium Sf9 insect cells (ATCC CRL 1711). In front of that plate Shake later to mix the newly added solution. The plate is then Incubated for 5 hours at ° C. Five hours later, the transfection solution was Rate and add 1 ml of Grace's insect medium supplemented with 10% fetal bovine serum. I got it. The plate was returned to the incubator and continued to be cultured at 27 ° C. for 4 days. .   Four days later, the supernatants were collected and as described by Summers and Smith (supra). Then, a plaque assay was performed. As a change, "Blue Gal" (Life Techno logies Inc., Gaithersburg). Thus, plaques stained blue can be easily isolated. (`` Plastic A detailed description of the `` Work Assay '' also provides a user's guide to insect cell culture, And Baculoy distributed by Life Technologies Inc., Gaithersburg Luthology, pp. 9-10).   Four days after adding serial dilutions of virus to the cells, blue stained pullers The sample was taken with the tip of an Eppendorf pipette. Then contains the recombinant virus The agar was resuspended in an Eppendorf tube containing 200 μl of Grace's medium. The agar is removed by brief centrifugation and contains the recombinant baculovirus. The supernatant was used to infect insect Sf9 cells seeded on 35 mm dishes. 4 days later The supernatants of these culture dishes were collected and stored at 4 ° C.   Sf9 cells were grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells are transformed at a multiplicity of infection (MOI) of 2 with recombinant baculovirus V-Ckβ-10. Infected. Six hours later, the medium was removed and methionine and cysteine were removed. Replace with SF900 II medium without media (Life Technologies Inc., Gaithersburg) I got it. 42 hours later, 5 μCi³⁵S-methionine and 5 μCi³⁵S Sistay 5 μCi (Amersham) was added. The cells were incubated for a further 16 hours Later, they were collected by centrifugation and the labeled protein was purified by SDS-PAGE. And visualized by autoradiography.   Since many modifications and variations of the present invention are possible in light of the above teachings, The invention may be practiced otherwise than as specifically described, within the scope of the following claims. .

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 48/00 ACD A61K 48/00 ADA ADA ADT ADT ADW ADW C07K 14/52 C07K 14/52 C12P 21/02 C C12P 21/02 A61K 37/02 ADU // (C12P 21/02 C12R 1:91)

Claims (1)

[Claims]   1. (A) a Ckβ-4 polypeptide having the deduced amino acid sequence of FIG. 1; Is a polynucleotide encoding a fragment, analog or derivative of the polypeptide. Nucleotides; (B) encoded by the cDNA contained in ATCC Deposit No. 75848 Ckβ-4 polypeptide having an amino acid sequence, or a flag of the polypeptide A polynucleotide encoding a comment, analog or derivative; (C) a Ckβ-10 polypeptide having the deduced amino acid sequence of FIG. Polynucleotides encoding polypeptide fragments, analogs or derivatives Otide; and (D) encoded by the cDNA contained in ATCC Deposit No. 75849 A Ckβ-10 polypeptide having an amino acid sequence, or a fragment of the polypeptide; A polynucleotide encoding a fragment, analog or derivative; An isolated polynucleotide selected from the group consisting of:   2. The polynucleotide according to claim 1, wherein the polynucleotide is DNA.   3. The polynucleotide according to claim 1, wherein the polynucleotide is RNA.   4. The polynucleotide according to claim 1, wherein the polynucleotide is genomic DNA. Chid.   5. The polynucleotide comprises Ckβ-4 having the deduced amino acid sequence of FIG. 3. The polynucleotide of claim 2, which encodes.   6. The polynucleotide comprises a Ckβ-10 having the deduced amino acid sequence of FIG. The polynucleotide according to claim 2, which encodes:   7. The polynucleotide is derived from the cDNA of ATCC Deposit No. 75848. The polynucleotide of claim 2, which encodes the encoded Ckβ-4 polypeptide. Creotide.   8. The polynucleotide is derived from the cDNA of ATCC Deposit No. 75849. 3. The polypeptide of claim 2, which encodes the encoded Ckβ-10 polypeptide. nucleotide.   9. 2. The polynucleotide according to claim 1, having the coding sequence of Ckβ-4 shown in FIG. Creotide.   10. 2. The polypeptide according to claim 1, which has the coding sequence of Ckβ-10 shown in FIG. Renucleotide.   11. Code of Ckβ-4 deposited under ATCC Deposit No. 75848 3. The polynucleotide of claim 2 having a sequence.   12. Coding of Ckβ-10 deposited as ATCC Deposit No. 75849 The polynucleotide according to claim 2, which has a nucleotide sequence.   13. A vector comprising the DNA according to claim 2.   14． A host cell genetically engineered with the vector of claim 13.   15. A method for producing a polypeptide, comprising the steps of: A method comprising expressing a repeptide from the host cell of claim 14. .   16. A method for producing a cell capable of expressing a polypeptide, A method comprising genetically engineering a cell with the vector of claim 13.   17． It is possible to hybridize to the DNA according to claim 2, An isolated DNA encoding a polypeptide having kβ-4 activity.   18. It is possible to hybridize to the DNA according to claim 2, An isolated DNA encoding a polypeptide having kβ-10 activity.   19. (I) a Ckβ-4 polypeptide having the deduced amino acid sequence of FIG. And fragments, analogs and derivatives thereof; (Ii) Ckβ-4 encoded by the cDNA of ATCC Deposit No. 75848 A polypeptide, and fragments, analogs and derivatives of the polypeptide; (Iii) Ckβ-10 polypeptide having the deduced amino acid sequence of FIG. Fragments, analogs and derivatives of (Iv) Ckβ-1 encoded by the cDNA of ATCC Deposit No. 75849 Polypeptides and fragments, analogs and derivatives of the polypeptides ; A polypeptide selected from the group consisting of:   20. The polypeptide is Ckβ-4 having the deduced amino acid sequence of FIG. The polypeptide of claim 19.   21. The polypeptide is Ckβ-10 having the deduced amino acid sequence of FIG. 20. The polypeptide of claim 19, wherein   22. An antibody against the polypeptide of claim 19.   23. An antagonist to the polypeptide of claim 19.   24. A method of treating a patient in need of Ckβ-4, comprising a therapeutically effective amount. 20. A method comprising administering the polypeptide of claim 19 to a patient.   25. A method of treating a patient in need of inhibiting Ck [beta] -4, the method comprising: Administering to the patient an effective amount of the antagonist of claim 23. Method.   26. A method of treating a patient in need of Ckβ-10, comprising treating the subject with a therapeutically effective A method comprising administering to a patient an amount of the polypeptide of claim 19.   27. A method of treating a patient in need of inhibiting Ckβ-10, comprising treating Administering an effective amount of the antagonist of claim 23 to the patient. Way.   28. 20. Any one of the polypeptides of claim 19, and pharmaceutically acceptable A pharmaceutical composition comprising a carrier that can be used.   29. A DNA encoding the polypeptide is provided to a patient, and the polypeptide is Expression in vivo results in the administration of a therapeutically effective amount of the polypeptide. 25. The method of claim 24, wherein   30. A DNA encoding the polypeptide is provided to a patient, and the polypeptide is Expression in vivo results in the administration of a therapeutically effective amount of the polypeptide. 27. The method of claim 26, wherein