JPH11508135A - Human EDG-2 receptor homolog - Google Patents

Abstract

(57) Abstract The present invention identifies a novel EDG-2 receptor homolog (hedg) expressed in human rheumatoid synovium and provides a nucleic acid and amino acid sequence encoding the same. The invention also provides probes for the detection of nucleotide sequences encoding HEDG or HEDG-like molecules, antisense molecules to nucleotide sequences encoding HEDG, diagnostic tests based on nucleic acid molecules encoding HEDG, for the production of purified HEDG. The present invention also provides an expression vector and a host cell which have been subjected to the genetic engineering treatment described above, an antibody capable of specifically binding to HEDG, and an antagonist and an inhibitor having a specific binding activity to the polypeptide HEDG.

Description

DETAILED DESCRIPTION OF THE INVENTION                       Human EDG-2 receptor homolog                                 Technical field   The present invention belongs to the field of molecular biology, and in particular, the present invention relates to a novel human EDG-2 receptor It describes the nucleic acid sequence and amino acid sequence of a homolog.                                 Background art   The EDG-2 receptor is a putative G clone originally cloned from sheep mRNA. It is a protein-bound 7 transmembrane receptor (T7G) (GenBank U1840) 5, Masana MI et al (1994) unpublished d). Human edg-1 is generally known because its endogenous ligand is unknown. Are classified as orphan receptors (Hla T and Maciag T ( 1990) J Biol Chem 265: 9308-13). T7G reception Some of the body has been classified as an orphan receptor. For such T7G receptor Is LCR-1 obtained from the brain, a mas oncogene involved in epidermoid carcinoma, RDC-1 known from several tumor tissues, and rat brain and testis And R334 obtained from Some of these have new ligands Some have been proposed for some time and have since been revoked. Orphan receptor Is the number of amino acids, molecular weight, glycosylation sites, and the presence of disulfide bonds. There are a variety of numbers (W atson S and Arkinstall S (1994) The G -Protein Linked Receptor Factors Book,   Academic Press, San Diego CA).   However, its seven hydrophobic groups traverse the plasma membrane to form a bundle of antiparallel α-helices Due to the sex domain, they are also closely related to other T7Gs. These transmembrane The segment (TMS) is represented by I-VII and describes the structure and function of the receptor. The characteristic features will be described. In many cases, a bundle of helices forms a binding pocket, If the binding site must be compatible with a bulkier molecule, extracellular N-terminal One or more of the end segments or the three extracellular loops Involved in the induction of conformational changes in the intracellular portion of the receptor after ovulation . The activated receptor interacts with an intracellular G protein complex, Are further involved in intracellular signal transduction activities (generally cyclic AMP (cAMP), Like follipase C, inositol triphosphate, or ion channel proteins Production of secondary messengers).   T7G receptors are expressed and activated in many developmental and disease processes Is done. Diagnosis of such processes by identifying novel T7G receptors Or the opportunity to intervene in it, and this receptor triggers its activity A physiological molecule or drug that prolongs or inhibits the duration of activity It can be used in screening assays for the identification of agent molecules.                                Disclosure of the invention   The present invention provides a novel human EDG-2 receptor homolog (HEDG). Provide a specifically encoding nucleotide sequence. Represented by hedg in this specification The cDNA obtained from Incyte Clone No. Using 8053, rheumatism Was identified and cloned from a synovial membrane cDNA library.   The present invention relates to activated, inflamed or related to the expression of HEDG. HEGD or its use in diagnosis or treatment of diseased cells and / or tissues Related to the use of variant nucleic acid and amino acid sequences. Embodiments of the present invention include he Cloning vector or expression vector containing dg antisense DNA, HEDG A host cell or organism transformed with an expression vector comprising HEDG, Method for producing and recovering purified HEDG from host cells, and signal involving HEDG Can be used to identify inhibitors of down-regulation of null transmission Purified protein HEDG                           BRIEF DESCRIPTION OF THE FIGURES   FIGS. 1A and 1B show the nucleic acid sequence of HEDG (coding region of SEQ ID NO: 1). FIG. 4 shows an alignment of the amino acid sequence (SEQ ID NO: 2). Array A Licensed by McCandis Software (Hitachi Software Engineer (Ring, Inc.).   FIG. 2 shows HEDG and sheep EDG-2 (U18405, SEQ ID NO: : 3) and human EDG-1 (GI119130, SEQ ID NO: 4) It is an event. Conserved Arg36 and Ser37 cuts unique to these T7G molecules Note the cut site. The sequence alignment of FIG. multis of tware (DNAStar Inc, Madison WI) equipment ali It was created using the Gnent program.                           Embodiment of the Invention   As represented herein by the uppercase abbreviation HEDG, SEQ ID NO: EDG2 receptor homologue in either naturally occurring or synthetic form having a two amino acid sequence And their fragments. In one embodiment, the polypeptide HEDG is , MRNA transcribed from the cDNA of SEQ ID NO: 1 (represented by the lowercase abbreviation hedg). ).   The novel human EDG-2 receptor homolog HEDG of the present application is a rheumatoid synovial The partial cDNA sequence expressed in the library (Clone No. 80 853). It is cloned from human vascular endothelial cells Expressed in epithelioid cells, fibroblasts, melanocytes, and vascular smooth muscle cells Has more homology to human edg-1.   "Oligonucleotide" is used in the polymerase chain reaction (PCR) method Sufficient bases to use as oligomers, amplimers, or probes It is a stretch of nucleotide residues. Oligonucleotides can be Or similar DNA in a particular cell or tissue prepared from a cDNA sequence. Or used to amplify, elucidate or confirm the presence of RNA. Oligonuk Reotides or oligomers can have a minimum of about 10 nucleotides and a maximum of about 30 nucleotides. , Preferably a portion of the DNA sequence having a length of about 25 nucleotides.   “Probes” refer to naturally occurring or recombinant single-stranded or Japanese-stranded nucleic acids. It can be derived or chemically synthesized. This probe can be the same or Useful in detecting the presence of similar sequences.   A "portion" or "fragment" of a polynucleotide or nucleic acid is about 6 kb. Less than nucleotides, preferably less than about 1 kb, which can be used as probes. And all or part of the nucleotide sequence. Such probes are Method, Klenow-fil-in reaction, PCR or other methods It can be labeled with a reporter molecule using known methods. Optimize the reaction conditions for false positive After a preliminary test to remove the sexuality, the DNA or R To determine whether NA is present in one cell type, tissue, or organ Southern hybridization, Northern hybridization, or in   Nucleic acid probes can be used in situ hybridization.   "Reporter" molecules are radionuclides, enzymes, fluorescent agents, chemiluminescent agents, Or a molecule to which a chromogen is attached, and has a specific nucleotide sequence or amino acid It is used to confirm the presence of the sequence and may allow its quantification. You.   "Recombinant nucleotide variants" encoding HEDG are based on the "redundancy" of the genetic code. The specific cleavage site and codon usage specific sudden By introducing various codon substitutions, such as silent changes that create mutations, Cloning into Rasmid or viral vectors, or specific prokaryotic or eukaryotic The expression in each of the cell lines can be optimized.   A “chimeric” molecule is a molecule that includes all or part of a nucleotide sequence of the present invention in an additional The following characteristics of HEDG are synthesized by introduction into a vector having an acid sequence. (Or more than one). The above characteristics include cell location, distribution , Ligand binding affinity, interchain affinity, denaturation / turnover speed Degree, signal transmission, etc.   The term “activity” refers to the biological and / or antigenic nature of any naturally occurring HEDG Form, fragment, or form of any HEDG polypeptide that retains the activity of Means main.   "Spontaneous HEDG" is produced by cells that have not been bioengineered Polypeptide, including, but not limited to, acetyl Including glycation, carboxylation, glycosylation, phosphorylation, lipidation and acylation Refers to various polypeptides resulting from post-translational modifications of the peptide.   The term “derivative” refers to ubiquitination, labeling (see above), (Derivatization with polyethylene glycol) and human proteins Like chemical insertion or substitution of amino acids like ornithine that are not normally produced Polypeptide that has been chemically modified by various techniques.   The term “recombinant polypeptide variant” is created using recombinant DNA technology. Differs from naturally occurring HEDG by the insertion, removal and / or substitution of amino acids Means any polypeptide that has become Impairs the activity of interest To determine amino acid residues that can be substituted, added or removed without knowing Compare the sequence of EDG with the sequence of closely related polypeptides to find highly conserved Any change in the amino acid sequence that occurs in a particular region may be targeted.   For "substitution" of an amino acid, for example, a leucine to isoleucine or valine Substitution, aspirate to glutamate, threonine to serine A single amino acid is similar in its structural and / or chemical properties to Is conservative when generated by substitution with a single amino acid.   “Insertions” or “deletions” typically range from about 1 to 5 amino acids Made in. This possible mutation can create synthetic peptides or use recombinant DNA technology. Systematically insert, remove, or replace nucleotides in the hedg sequence It can be determined empirically by sizing.   "Signal or leader sequences" can be added to the polypeptide through the cell membrane, if necessary. Can be used to give instructions to the code. Such a sequence is a polypeptide of the invention. Heterologous polypeptides, either naturally occurring on Source.   An “oligopeptide” is a short stretch of amino acid residues that It can be expressed from leotide. This oligopeptide is a polypeptide Functionally equivalent and identical to “fragment”, “part” or “segment” It can be as long (or significantly shorter). Such an array is at least Also about 5 amino acids, often about 17 or more amino acids, typically at least Consists of about 9 to 13 amino acids and is sufficient to exhibit biological and / or antigenic activity. Includes stretches of amino acid residues of variable length.   "Inhibitors" delay or inhibit chemical or physiological reactions Any substance. Common inhibitors include antisense molecules, antibodies, And antagonists, but are not limited to these.   “Standard” expression is used as a reference in quantitative or qualitative measurements. is there. It is based on a statistically relevant number of routine sources, To run, conduct clinical trials, or follow a patient's treatment profile Created for use as a basis for comparisons.   As used herein, the term “animal” refers to humans, companion animals (cats, dogs, etc.), Includes livestock (cows, horses, sheep, etc.) or experimental animals (mouse, rat, rabbit, etc.) Can be defined as   The present invention uniquely describes a novel seven transmembrane receptor, human EDG-2 or HEDG. Provide the nucleotide sequence to be identified. HEDG in the inflammatory rheumatoid synovium Nucleic acid (hedg), polypeptide (HEDG) and And hedg are diagnostic assays to investigate increased receptor production It is useful in. Overexpression of HEDG is associated with T cells and other cells that respond to inflammation. Often associated with vesicle activation, causing large amounts of proteases and tissue damage or destruction This may result in the production of other molecules that can be linked. Therefore, the excessive occurrence of HEDG Diagnostic tests of the present include viral, bacterial or fungal infections, allergic reactions, Severe injury due to trauma, genetic disease, lymphoma or carcinoma, or inheritance of lymphoid tissue Diagnosis and appropriate treatment of abnormal conditions caused by other conditions that activate the child It can promote.   The nucleotide sequence encoding HEDG (or its complementary sequence) It has many uses in techniques well known to those skilled in the field of physics. this These techniques include use as hybridization probes, PCR oligos. Use as a marker, use in chromosome and gene mapping, HEDG recombination Use in the production of rodents, and antisense DNA or RNA, and their chemistry And the like in the production of statistical analogs and the like. However, HEDG disclosed here is The use of the encoding nucleotide sequence is merely an example of known technology and will It is not intended to limit use in any of the known techniques. Furthermore, it is still open Unpublished molecular biological techniques, such as triplet genetic Based on known polynucleotide sequence characteristics, such as signal and specific base pairing interactions The nucleotide sequence disclosed herein can be used in any molecular biological technique. Can be used in surgery.   As a result of the degeneracy of the genetic code, a variety of HEDG encoding Can be generated. Some of them are known nucleoti Sequence and minimal homology to the nucleotide sequence of naturally occurring HEDG Some have a nucleotide sequence of The invention more specifically describes the possible All possible nucleons that can be created by selecting combinations based on Pseudo sequences are included in the range. These combinations are based on the naturally occurring HEDG Based on the standard triplet genetic code as applied to leotide sequences Formed. Also, all such variants are specifically described herein. I want to be considered.   The nucleotide sequence encoding HEDG and / or HEDG variants may be strict Hybridizable with naturally occurring hedg nucleotide sequences under mild conditions HED processing substantially different codon usage Creating a nucleotide sequence encoding a G derivative or HEDG is beneficial. possible. Codon selection involves specific prokaryotic or eukaryotic expression hosts. It can be chosen to increase the rate of expression of the peptide, where the rate of expression is Is determined based on the frequency of use of a particular codon in the host. HE of the present invention The nucleotide sequence encoding a DG and / or HEDG derivative is Other reasons for substantially changing the amino acid sequence without altering it are more desirable Characteristics, such as longer half-lives than those formed from naturally occurring nucleotide sequences In order to create an RNA transcript that   Nucleotide sequences encoding HEDG can be substituted by a fully established recombinant DNA technology. ("Sambrook J et al.   (1989) Molecular   Cloning: A Laboratory Manual, 2d Ed , Cold Spring Harbor, New York City " ) With various other nucleos It may bind to a peptide sequence. a useful nucleotide sequence to bind hedg For example, conventionally known plasmids, cosmids, lambda phage derivatives, phage Various cloning vectors such as mid are included. Vectors of interest include , Expression vector, replication vector, probe generation vector, and sequencing Vector and the like. Generally, at least one vector of interest Origin of replication, convenient restriction endonuclease detection sites, and one or more Or may include selectable markers for two or more host cells.   In another embodiment of the present invention, a naturally occurring nucleotide sequence encoding HEDG is Hybridizable hedg-specific nucleic acid hybridization probes Provided. Such probes detect similar HEDG encoding sequences. Preferably, the nucleotide sequence of the conserved region or active site is used. Contain at least 50% of the The hybridization probe of the present invention The nucleotide sequence of SEQ ID NO: 1, or the promoter of the naturally occurring hedg, It may be derived from a genomic sequence that includes a sensor element and an intron. C The hybridization probes can be generated by various reporter groups using well-known techniques. Can be labeled.   U.S. Pat. Nos. 4,683,195, 4,800,195 and 4,965 In performing a PCR method as described in US Pat. Alternative Uses of Oligonucleotides Based on Encoding Nucleotide Sequences . Probes used in such PCR are those obtained by recombination. It may be chemically synthesized, or a mixture of both. Oligo Optimized conditions for hedg identification and diagnostic use in specific tissues Including the individual nucleotide sequences used below. The same two oligomers, put Orient in child state Gomer pairs, or pools of degenerate sequences, are closely related It can be used under less stringent conditions for the identification of genomic sequences.   for generating hybridization probes specific to hedg Other methods include HEDG and H into vectors for the formation of mRNA probes. Cloning of nucleic acid sequences encoding EDG derivatives is included. Such a vector Are well known and commercially available, for example, T7 or SP6 RNA A suitable RNA polymerase such as a polymerase and a suitable radiolabel. RNA probes in vitro by adding the selected nucleotides Can be used to   At present, DNG encoding HEDG and HEDG derivatives is completely chemically synthesized. It is possible to generate the A sequence, or a part thereof. After synthesis, Various commercially available DNA vectors and their host cells can be prepared using known techniques. Can be inserted. In addition, using chemical synthesis, the nucleotide sequence or Can be mutated. Another form is array projection. The part that is susceptible to mutation is synthesized and is part of an existing genomic or recombinant sequence. It can be recombined with a longer one in part.   The nucleotide sequence of hedg is used to correlate abnormal levels of HEDG expression. Assays for the detection of inflammation and disease can be constructed. This cDNA Is labeled with a well-known method, and then the body of the patient is subjected to hybridization conditions. It can be added to a liquid, cell or tissue sample and incubated. After the incubation time has elapsed, if necessary, The sample is washed with a neutral liquid. After rinsing this compatible liquid, remove the reporter Quantify the child Compare with the set standard value. If the kinase expression was significantly different from the standard expression In this case, the assay confirmed the presence of inflammation and / or disease. You.   For mapping of the native gene using the nucleotide sequence of hedg Can be constructed. Nu to disclose here Mapping of nucleotide sequences to chromosomes and specific regions of chromosomes can be performed using well-known genetic It can also be performed using offspring and / or chromosome mapping techniques. Such techniques Surgery includes chromsome spread (Verma et al (198). 8) Human Chromosomes: A Manual of Ba sic Technologies, Pergamon Press, New York City), flow-sorted chromosomal preparations, yeast A bacterial artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a bacterial P1 construct, or In situ artificial chromosome synthesis, such as a single chromosomal cDNA library Hybridization is included.   In situ hybridization of chromosome preparations and established chromosomes Physical mapping techniques such as linkage analysis using markers, extended gene sites It is not visible in the figure. As an example of genetic map data, "1 994 Genome Issue of Science (265: 1981). f) ". By determining the location of a gene on the chromosome of another mammal, Of linked markers that can be used to help identify human chromosomes Often confirmed.   New nucleotide sequences are assigned to small regions of the chromosome by physical mapping. Can be done. New genes or nucleotide sequences can be mapped to position Disease genes using cloning and other gene discovery techniques Useful landmarks can be obtained for researchers searching for. Once ataxia telangiectasia (AT ) Are linked to a specific region of the genome (eg, AT To 11q22-23 (Gatti et al (1988) Natu re 336: 577-580)). Sequence mapping expresses and confirms genes for further investigation It will be. Using the nucleotide sequence of the present invention, a gene sequence of a healthy subject and Differences from the gene sequence of a carrier for a hereditary or hereditary disease can also be detected.   The nucleotide sequence encoding hedg can be modified using well known recombinant DNA techniques. Can be used to create purified oligopeptides or polypeptides . There are many literatures describing methods for expressing a gene after its isolation. However, as an example, “Goeddel (1990) Gene Express session Technology, Methods and Enzymo logic.   Vol 185, Academic Press, San D iego CA ". This oligopeptide is useful for prokaryotic or eukaryotic cells. These can be expressed in a variety of host cells. The host cell has the nucleotide sequence Can be obtained from either the same species that originates from, or from different species. set The advantages of producing oligonucleotides by recombinant DNA technology include purification To obtain a sufficient amount of protein and simple procedures for purification. May be available.   Cells transformed with DNA encoding HEDG are T7G, its cells. Expression of extracellular transmembrane or intracellular region and recovery of protein from cell culture Can be cultured under appropriate conditions. HEDG produced by recombinant cells (also , Any of its regions) may be secreted or Alternatively, it can be retained intracellularly. General In addition, it is more convenient to prepare the recombinant protein in a secreted form. The purification step depends on the nature of the production process used and the particular protein produced. Determined based on the nature of the material. In many cases, the oligopeptide is a chimeric nucleo It can be produced from a peptide sequence. This is a polypeptide that promotes protein production A nucleic acid sequence encoding main and nucleotides from hedg or this polypeptide. By combining the desired portion of the peptide (Kroll DJ et al (1993) DNA Cell Biol 12: 441-53).   In addition to recombinant production, direct peptide synthesis using solid-phase technology G fragments can also be produced. ("Stewart et al ( 1969) Solid-Phase Peptide Synthesis,   WH Freeman Co.   San Francisco; Merri field R (1963) J Am Chem Soc 85: 2149 -2154 "). For automatic synthesis, see, for example, Applied Biosystem. ems 431A Peptide Synthesizer (Foster City, CA) according to the manufacturer's instructions. You. In addition, certain parts of HEDG can be mutated during direct synthesis. Alternatively, it can be linked to other parts of the peptide using chemical methods.   HEDG for use in eliciting antibodies must have biological activity. Although not required, the protein must be antigenic. HEDG-specific antibodies At least 5, preferably at least 1, It may include an amino acid sequence consisting of 0 amino acids. This peptide is It should be identical to a part of the amino acid sequence of the protein, All amino acids in naturally occurring molecules May contain columns. The antigenic portion of HEDG is the chimney hemocyanin (K LH) and other proteins such as chimeric molecules used for antibody production. You.   Antibodies specific for HEDG can be used in appropriate animals to express polypeptides or their antigenic antibodies. It can be produced by inoculating a fragment. The antibody is an epitope of the polypeptide Produced at least part of naturally occurring or recombinant proteins If so, the antibody is said to be specific for HEDG. Antibody production Life is not only due to the stimulatory effect of the immune response caused by injection into animals, but also Screen for specific binding molecules from adult antibodies and recombinant immunoglobulin libraries ("Orlandi et al (1989) PNAS 86: 3833- 3837 or Huse et al (1989) Science 256 In vitro stimulation of lymphocyte populations. It also happens by. Current technology (“Winter and Milstein” (1991) Nature 349: 293-299 "). Based on the theory, a large number of highly specific binding reagents can be provided. this Such techniques can be applied to the production of molecules that can specifically bind to HEDG. You.   In yet another embodiment of the invention, HEDG-specific antibodies, inhibitors, receptors Or its analogues as a bioactive agent for the treatment of inflammation and disease. You. These diseases include viral, bacterial, or fungal infections, allergic reactions, and trauma. Related mechanical damage, genetic disease, lymphoma or carcinoma, or There are other conditions that may be included, but are not limited to:   HEDG agonists, antagonists, receptors, or inhibitors The bioactive composition comprises a mammal for determining the maximum tolerated dose. Includes clinical studies in humans and routine human studies to determine safe doses Can be administered at an appropriate therapeutic dose as determined by any of several methodologies . In addition, the bioactive agent enhances pharmacological properties such as stability or half-life. Can be synthesized with a variety of well-established compounds or compositions. Also this Therapeutic bioactive compositions can be delivered to the bloodstream by intravenous injection or By other effective means that can be used to treat problems involving papocytes and leukocyte transport It is contemplated that it can be administered.   Rheumatoid arthritis is now based on swelling, NSAID response, X-rays, etc. Is evaluated. HEDG is often expressed in fibroblasts, T cells and And B cells. Mast cells, including monocytes / macrophages, or inflammatory synovial cells Surface. Once an appropriate standard value has been established, the abnormal expression of HEDG Say is a biodiagnostic tool to assess the degree of progression of rheumatoid arthritis. The expression of HEDG in the sustained inflammatory response makes it possible for the drug library It is a therapeutic target useful for cleaning. HEDG inhibitors are rheumatism Helps to regulate signaling and signaling cascades in synovial cells.   The following examples are provided to illustrate the invention. These examples are , Are intended to be illustrative, and not limiting, of the present invention.                             Industrial applicability 1.mRNA isolation and cDNA library construction   The Hedg sequence is an insight in the sequence group including the rheumatoid synovial membrane library. Clone No. First identified from 80853. Rheumatism Synovial tissue was removed from a 68-year-old woman with erosive rheumatoid arthritis Obtained from the verse. This tissue is frozen, crushed in a mortar and pestle, and It was dissolved in a buffer containing diisothiocyanate. For this solution Then, phenol-chloroform extraction and ethanol precipitation were performed several times. Poly A + mRNA bound to biotinylated oligo d (T) and paramagnetic particles Isolated using Streptavidin (Poly (A) Tact Iso ration System, Promega, Madison WI).   Using this poly A + mRNA, a custom-made cDNA library Constructed by Tratagene (La Jolla CA). cDN Synthesis of A is primed with oligo d (T) and the adapter oligonucleotide Is linked to a cDNA molecule, which is then transferred to the Uni-ZAP® vector system. System (Stratagene). Another one Directional vectors include pcDNAI (Invitrogen, San Dieg). o CA) and pSHIox-1 (Novagen, Madison WI) But not limited to them. 2.Isolation of cDNA clones   The phagemid form of the cDNA clones here is based on the in vivo excision process. Obtained by. In this process, the host strain contains the library phage and f1 Both helper phages were co-infected. Library-containing phages and helpers -Polypeptides or enzymes from both phages can cut into DNA. To initiate new DNA synthesis from the defined sequence on the target DNA the entire DNA sequence of the euscript phagemid and cDNA insert A smaller single stranded circular phagemid DNA molecule was generated. This fur Zimide D NA is released from the cells and produced, producing new host cells (SOLR®). (Stratagene). In this host cell 2 Single-stranded DNA was produced.   DNA was purified using the QIAWELL-8 plasmid purification system (QIAGEN Inc.). , Chatsworth CA) and EMPORE (R) membran e Anion exchange resin system with technology (3M, Min Neaporis MN). This DNA is dissolved from the purified resin. Separated and prepared for DNA sequencing and other analytical procedures. 3.Sequencing of cDNA clones   CDNA insulators randomly isolated from these rheumatoid synovial libraries The plate was partially sequenced. In conventional methods using enzymes, DNA Rase Klenow Fragment SEQUENASE® (US Bioc) chemical Corp. Cleveland, OH) or Taq polymer Annealed to a DNA template of interest using an enzyme such as Rase Extend the DNA strand from the oligonucleotide primer. Such a method is one Developed for use with both single-stranded and double-stranded templates. The product of the chain termination reaction is Separated by electrophoresis and urea acrylamide gel, autoradiograph Method (when using a precursor labeled with a radionuclide), or a fluorescent agent (a fluorescent agent) (When using a precursor labeled with). Reaction preparation, distribution Determined in one day due to recent mechanization in sequencing and analysis using fluorescence detection The number of arrays to be used can be increased. The machine used is th e Catalyst 800 and the Applied Biosystem ems 377 and 373 DNA sequencers. 4.Homology search for cDNA clones and deduced proteins   Each cDNA is a search algorithm manufactured by Applied Biosystems Was incorporated into the INHERIT ™ 670 sequence analysis system and used to Comparison was performed with the sequence of nBank. In this algorithm, Pattern n Specification Language (manufactured by TRW, Los (Angles CA) was used to determine the homologous region. What is sequence comparison The three parameters that determine how to do this are window size and window offset. And error tolerance. Using a combination of these three parameters, Search the DNA database for sequences containing regions with homology to the title sequence The appropriate array was scored with initial values. Subsequently, these homologs The regions were tested using the dot matrix homology plot method and the chance matches and true A homologous region was distinguished. Smith-Water to display the results of the homology search rman alignment was used.   The homology of the peptide and protein sequences is based on the INHERIT ™ 670 sequence Confirmation was performed in a manner similar to DNA sequence homology testing using an analytical system. Pa ttern Specification Language and Parameter Using a window, search a database of protein sequences containing regions of homology, Homologous regions were scored and displayed with initial values. Dot matrix homology A test was performed by the sex plot method to distinguish significant homology regions from accidental matches.   Alternatively, BLAST (Basic Local Match Search Tool) (“Alts chul SF (1993) J Mol Evol 36: 290-300 Altschul, SF et al (1990) J Mol Bi; 215: 403-10 ") for local sequence matches. . BLAST consists of nucleotides and The identity of both amino acid sequences is checked to determine sequence similarity. Local of agreement Because of their unique nature, BLAST seeks exact matches or identifies homology. It is particularly useful for BLAST is useful for finding gap-free matches. It is for. The basic unit of the BLAST algorithm output is High-sc oring Segment Pair (HSP).   The HSP is arbitrarily equal in length, the match between each other is locally maximal, and the match score is User meets or exceeds the cut-off or threshold score set by the user Consists of two sequence fragments. By the method using BLAST, Search for HSPs between the sequence in question and the database sequence and find the statistical You can evaluate the significance and know only those matches that meet the significance threshold selected by the user. Wear. Parameter E is used to select the one reported on the match with the database sequence. Set a statistical significance threshold for E is the context of the entire database search Expected upper frequency limit of chance coincidence of HSP (or set of HSPs) Is done. Database sequences that satisfy E are reported in the output of the program. 5.Identification, full-length cloning, sequencing, and translation   Randomly caught and sequenced from a rheumatoid synovial library As a result of analyzing part of the loan using INHERIT ™, sheep EDG-2 As a homologue of the receptor, a clone No. 80853 was identified. This partial sequence corresponds to the nucleotide sequence of GenBank inventory U18405 and 9 2.6% match (Masana MI et al, supra). Insight Clone No. The cDNA insert containing 80853 was completely sequenced. Used as a basis for cloning full-length cDNA.   This cDNA was obtained from Guegler et al, filed June 7, 1995. . Modified XL-P described in U.S. patent application Ser. No. 08 / 487,112, Using the CR (Perkin Elmer) procedure, the rheumatoid synovial It was extended to full length using the slurry as a template. Primer is a known sequence Designed based on One of the primers is in the antisense direction (XLR = TCA TCTTGATCCCCATCCCTTCTG) to initiate extension And the other is sensed (XLF = AGTCTCCCGAGTATTG GGTCCTGTG) to extend the sequence. This plastic Immers extend the sequence "outward", creating new unknowns for the gene of interest Can be generated. This plastic The immer was oligo 4.0 (National Bioscience Inc, (Plymouth MN) and has a length of 22 to 30 nucleotides. It has a GC content of 50% or more and has a target distribution at a temperature of about 68 to 72 ° C. It can be designed to anneal to the rows. Primer-primer with hairpin structure Avoid generation of stretches of arbitrary nucleotides that cause dimer formation Is done.   Mix the enzyme and the reaction mixture thoroughly according to the instructions of the XL-PCR kit. This achieves high fidelity amplification. 25 pMol of each primer and PCR started with all other components of the kit at the recommended concentrations was performed using MJ   For PTC200 (MJ Research, Watertown MA) And with the following parameters: Step 1 at 94 ° C for 60 seconds (initial denaturation) Step 2 15 seconds at 94 ° C Step 3 at 65 ° C for 1 minute Step 4 7 minutes at 68 ° C Step 5 Repeat steps 2 to 4 15 more times Step 6 at 94 ° C for 15 seconds Step 7 at 65 ° C for 1 minute Step 8 7 minutes at 68 ° C + 15 seconds / cycle Step 9 Repeat steps 6 to 8 11 more times Step 10 8 minutes at 72 ° C Step 114 4 ° C (keep this temperature)   At the end of cycle 28, 50 μl of the mixed reaction was removed and the remaining reaction An additional 10 cycles are performed on the mixture. The outline is shown below. Step 1 15 seconds at 94 ° C Step 2 at 65 ° C for 1 minute Step 3 at 68 ° C. (10 minutes + 15 seconds) / cycle Step 4 Repeat steps 1 to 3 9 more times Step 5 10 minutes at 72 ° C   A 5-10 μl aliquot of the reaction mixture was analyzed on the minigel and the reaction was successful. It was decided whether or not. Any extension reaction products may contain full-length genes But the largest product is selected and low concentration (about 0.6-0.8%) agaro Separated from the template by electrophoresis on a gel. Remains of purpose The band representing the gene was excised from the gel and the QIAQuick ™ gel Extraction kit (QIAGEN Inc, Chatsworth CA) Produced using the method. Using Klenow enzyme, overhang the end Were trimmed to blunt ends for easy ligation and cloning.   After ethanol precipitation, the product was redissolved in 13 μl of ligation buffer. One 1 μl of T4-DNA ligase (15 units) and 1 μl of T4 polynucleotide Otide kinase is added and the mixture is allowed to stand at room temperature for 2-3 hours or at 16 ° C. Incubated overnight. Competent E. coli cells (40μ) l suitable medium) are transformed with 3 μl of the ligation mixture and 80 μl of SOC Cultured in medium (Sambrook J et al., Supra). 37 ° C After incubation for 1 hour at room temperature, all of the transformation mixture was washed with 25 mg / L Luria Bertani (LB) -agar containing lubenicillin (Sam brook, J., supra (Sambrook J., supra) ) Was plated. At a later date, 12 colonies were randomized from each plate. Each well of an appropriate commercially available sterile 96-well microtiter plate. And cultured in 150 μl of liquid LB / carbenicillin medium. Cultured overnight at a later date Each 5 μl was transferred to a non-sterile 96-well plate, diluted 1:10 with water, and then Were transferred to a PCR array.   For amplification by PCR, 0.75 units of Taq polymerase vector plasmid One or more gene-specific primers used for the immersion and elongation reactions 15 μl of the enriched PCR reaction mixture (1.33 ×) containing was added to each well. Increase The width was performed under the following conditions. Step 1 94 ° C for 60 seconds Step 2 at 94 ° C for 20 seconds Step 3 55 ° C for 30 seconds Step 4 at 72 ° C for 90 seconds Step 5 Repeat steps 2 to 4 29 more times Step 6 180 seconds at 72 ° C Step 7 4 ° C (Keep the temperature)   Aliquots of the PCR reaction were run on agarose gels along with molecular weight markers. I responded. Compare the size of the PCR product with the original partial cDNA and select the appropriate clone. Was selected and ligated to the plasmid and sequenced.   The nucleotide and amino acid sequences of human HEDG are shown in FIG. The coding region of hedg begins at nucleotide 309 of SEQ ID NO: 1. Ends at nucleotide 1403. Possible translation of HEDG, Swiss Searching protein databases such as Prot and PIR Nothing was found. FIG. 2 shows the HEDG sequence and the sheep EDG. -2 (U18405) and human EDG-1 (GI119130) sequences It is. 6.Antisense analysis   Knowing the correct and complete cDNA sequence of HEDG enables investigation of gene function This can be applied to antisense technology in the field. Hedg Ann Oligonucleotide, cDNA or genomic fragment containing the Inhibiting mRNA expression using in vitro or in vivo Can be. Such techniques are well known and attach to various sites in the nucleotide sequence. Antisense molecules can be designed. Whole cells or laboratory animals Effectively block gene function by treating with antisense sequences such as Can be Often, at the cellular, tissue, or whole organism level Observe behavior at the level (eg mortality, loss of differentiated function, morphological changes, etc.) By doing so, the function of the gene can be confirmed.   In addition to using sequences constructed to prevent transcription of open reading frames, An intron region, a promoter / enhancer element, or By designing antisense sequences for trans-acting regulatory genes, Gene expression can be modified. Similarly, a triple helix (triple helix) Cux) achieves inhibition using Hogeboom base pairs known as "base pairs" be able to. 7.HEDG expression   Hedg expression is achieved by subcloning the cDNA into an appropriate expression vector. By introducing the vector into an appropriate expression host. Specific like this In the case of the cloning previously used for the generation of the cDNA library The vector is also E. direct expression of the hedg sequence in E. coli. Black Upstream of the cloning site, this vector contains the promoter for β-galactosidase. With an amino-terminal Met followed by 7 of β-galactosidase. Includes a sequence containing two residues. Immediately after these eight residues, an artificial priming and Bacteriophage promoters that have been engineered to It has a number of unique restriction sites for learning.   The isolated IPTG was transfected using standard methods. By induction of the bacterial species, the first seven residues of β-galactosidase and about “linker” Fusion protein corresponding to 15 residues, and peptide encoded by cDNA Is generated. cDNA clone inserts are always produced by a random process. The contained cDNA is in the correct reading frame for proper translation. There are only one in three opportunities. If the cDNA is not in the appropriate reading frame It can be obtained by deleting or inserting an appropriate number of bases in a well-known manner. Can be Such methods include in vitro mutagenesis, exonuclease Digestion with Rease III or soybean nuclease, or oligonucleoside Tide linker contamination and the like.   Hedg cDNAs are useful for expression of proteins in specific hosts. Shuttle into another vector known to be. The two ends of the target cDNA Enough segments of DNA (approximately 25 Oligonucleotide bases containing the cloning site It can be synthesized chemically by standard methods. Then, using these primers, The desired gene segment is amplified by PCR. New gene segment obtained Are digested with the appropriate restriction enzymes under standard conditions, and analyzed by gel electrophoresis. Isolated. Alternatively, similar gene segments can be replaced by cDNA Is produced by digesting with appropriate restriction enzymes. Use the appropriate primer The segments of the coding sequence from one or more genes Cloning with a unique vector. Expression is optimized by constructing such a chimeric sequence. It is possible to adapt.   Suitable expression hosts for such chimeric molecules include Chinese hamsters. Mammalian cells such as ovary (CHO) and human 293 cells, yeast cells, E. . bacteria, such as, but not limited to, E. coli. like this Expression vectors used for each of the various cell lines Origin of replication that allows for cloning, and β-lactamase antibiotics that allow for selection in bacteria It contains a selectable marker such as a quality resistance gene. Furthermore, this vector Is a neomycin phosphoprotein useful for transfection of eukaryotic host cells. Include a second selectable marker, such as a transferase gene. Eukaryote For use in an expression host, the vector may comprise a portion of the cDNA of interest. If not, an RNA processing element such as a 3 'polyadenylation sequence I need.   In addition, this vector contains an enhancer or promoter that increases gene expression. Including. Such promoters are host-specific and, for CHO cells, MM TV, SV40 and metallothionein promoters, trp for bacterial hosts , Lac, tac, and T7 promoters, and for yeast, α-factor, alcohol Oxidase, and PGH promoter. Rous sarcoma virus (RS V) Transcriptional enhancers, such as enhancers, are used in mammalian host cells . Once a homogenous culture of recombinant cells has been obtained using standard culture methods For example, a large amount of HEDG produced by recombination is recovered from the conditioned medium and It is analyzed using a chromatographic method. 8.Isolation of recombinant HEDG   HEDG is one or more additions added to facilitate protein purification. Expressed as a chimeric protein having a specific polypeptide domain. like this Histidine-triplets that allow purification on immobilized metals Metal chelating peptides such as Tophan modules, on fixed immunoglobulins Protein A domain allowing purification and FLAGS extension / affinity System (Immunex Corp. Seattle WA). Domain used, but is not limited to these. Purification Factor XA or enterokinase between the main and hedg sequences (Invtro gen) can include a cleavable linker sequence, Helps to facilitate purification. 9.Testing of chimeric T7G   Functional chimeric T7Gs are known to contain extracellular receptor sequences for new isoforms. Is constructed by transmembrane and binding to an intracellular segment of the isoform. Such chimeric molecules are useful for testing. This Is based on the idea of Kobilka (1988, Science 240: 1310). -1316), with higher amounts of α2-AR transmembrane sequences A series of chimeric α2-β2 adrenergic receptors by insertion into the β2-AR (AR). A conformation in which the molecule has more α2 than β2 The binding activity of known agonists changes when shifted from Construct mixed specificity. However, specificity and domain binding to antagonists There was a correlation with the source of InVII. T7G for ligand recognition The importance of domain VII is also found on chimeras using two yeast α-factor receptors However, this is because yeast receptors are classified as miscellaneous receptors. is important. Therefore, the functional role of a specific domain depends on its category. All appear to be conserved throughout the T7G family.   In parallel form, internal segments or cells from a particular isoform The cytoplasmic domain is exchanged for a known T7G analogous domain and the receptor and trimer G Used to identify structural determinants that associate with proteins (Dohlman   et al (1991) Annu Rev Biochem 60:65. 3-88). The intracellular binding loop from domains V, VI, and β2-AR is α2 -AR-substituted chimeric receptor specifically binds ligand and α2-AR However, it was found to stimulate adenylation cyclase like β2-AR . This suggests that the G protein recognition site is in a domain because of adrenergic receptors. In V and VI and their binding loops. Opposite state Is also predicted, and the V → VI loop from α1-AR has a corresponding domain on β2-AR. And the resulting receptor specifically binds the ligand to the β2-AR, -G-protein-dependent phospho similar to AR This was observed for chimeras that activated atidyl inositol turnover. Finally, chimeras constructed from the muscarinic receptor also have a V → VI loop with a G protein. It has been established that there is a major determinant for the specificity of cytoplasmic activity (Bolande) rFF, supra).   Chimeric or modified T7Gs containing substitutions in extracellular and transmembrane domains Showed that both parts of the receptor determined the ligand binding specificity. example For example, two Ser residues are adrenergic and D-catecholaminergic receptors. Conserved in all domain V of the body and essential for exerting agonist activity It is. These serines are located at the T7G binding site and are Forming hydrogen bonds that combine. Similarly, all T7G domains that bind to biogenic amines The Asp residue present in main III is located at the T7G binding site and It is believed to form an ion pair with the cation group.   Functionally, the cloned T7G is expressed in a heterologous expression system and Physical activity was evaluated (Marullo et al (1988) Pro c Natl Acad Sci 85: 7551-55; King et. al (1990) Science 250: 121-23). One of heterogeneous systems Introduces genes for mammalian T7G and mammalian G protein into yeast cells . This T7G shows that it has appropriate ligand specificity and affinity. Triggers the appropriate biological activation of yeast cells-growth arrest and morphological changes- It was shown to be.   Another procedure for testing chimeric receptors is described in Erb et al (1993,   Proc Natl Acad Sci 90: 10449-53). U, P_2UPurine receptor (P_2U)). Its function is Easily with substituted K562 human leukemia cells Tested, which indicates that these cells_2UThis is because they lack the receptor. K5 62 cells are normal or chimeric P_2UTransfer of an expression vector containing any of Action, Ca⁺⁺Loaded with a fluorescent probe, fura-a. Appropriate Functional P collected in_2UActivation of the receptor by extracellular UTP or ATP And intracellular Ca⁺⁺Moves, which reacts with fura-a and is measured spectrofluorometrically Is done.   In the presence of the T7G receptor described above, the chimeric gene may be any newly discovered T7 The sequence of the extracellular receptor segment of the G polypeptide and the known P_2UTransmembrane penetration of molecules And the sequence of the intracellular segment. Transfer The treated K562 cells are then transferred to a microwell (micr) containing the appropriate ligand. owell) to limit the effectors of T7G molecules by bathing Triggers binding and fluorescent activity. Once ligands and functions are established , P_2UThe system may be an inhibitor or inhibitor that blocks binding and prevents such fluorescent reactions. Will help define the antagonist. 10.Generation of HEDG-specific antibodies   Two methods are used to generate antibodies against HEDG. Each person Method is useful for generating either polyclonal or monoclonal antibodies It is something. In one of the methods, reverse phase HPLC separation allows the denaturing protein Is obtained up to 75 mg. Denatured proteins can be expressed in mice using standard protocols Or used to immunize rabbits. That is, about immunization of mice 100 μg is appropriate and up to 1 mg can be used for immunization of rabbits. Mau For identification of hybridomas, the denatured protein is labeled with radioactive iodine, For screening murine B cell hybridomas capable of producing antibodies I have. The procedure requires only a small amount of protein, i.e., thousands of 20 mg is sufficient for labeling and screening of the protein.   In the second method, the amino acid sequence of HEDG deduced from the transcription of cDNA is used. The rows are analyzed to determine areas of high immunogenicity. Contains appropriate hydrophilic regions Oligopeptides are synthesized and suitable immunization protocols for antibody production are used. Used. Analyzes to select appropriate epitopes are available from Ausubel. FM and the like (described above). Optimal amino acid sequence for immunization Usually, the protein exists between the C-terminus and the N-terminus and between them. Hydrophilicity of polypeptides often exposed to the external environment when in its natural conformation Sex region.   Typically, a selected peptide of length about 15 residues will have an fmoc -Applied Biosystems Pep using -chemistry synthesized using tide Synthesizer Model 431A, M-maleimidobenzoyl-N-hydroxysuccinimide ester (M-male Reaction with imidobenzoyl-N-hydroxysuccinimide ester) (MBS; Ausube) l FM, etc., supra) to allow keyhole limpet hemocyanin ( KLH, Sigma, St Louis MO). K if necessary Cysteine is inserted at the N-terminus of the peptide so that it can bind to LH . Rabbits were immunized with Freund's complete adjuvant peptide-KLH complex. Be transformed into The resulting antiserum combines the peptide with plastic and produces 1% bovine Block with serum albumin, react with antiserum, wash and (radioactive or Affinity-purified specific goat anti-rabbit IgG labeled with a fluorescent agent) The anti-peptide activity is tested by reacting with   Hybridomas are prepared and screened using standard techniques. You. The desired hybridoma is screened together with the labeled HEDG To produce a monoclonal antibody with the desired specificity. Coalescence is identified. In a typical protocol, plate wells (FAST;   Becton-Dickinson, Palo Alto, CA) 0 mg / ml of the affinity-purified specific rabbit anti-mouse (or Coated with anti-species Ig) antibody. 1% coated holes Blocked with BSA, washed and exposed to the supernatant from the hybridoma. After incubation, the wells are exposed to 1 mg / ml labeled HEDG. Clones producing antibodies are detected in amounts detectable in the background described above. Binds to labeled HEDG. Such clones are expanded and subjected to limiting dilution ( (1 cell / 3 wells) undergo 2 cycles of cloning. Hive cloned Redomas are injected into pristine-treated mice, which produce ascites and Monoclones from ascites of mice by affinity chromatography on Rotin A Clonal antibodies are generated. At least 10⁸/ M, preferably 10⁹-10^Ten Or higher affinity monoclonal antibodies are typically obtained from Harl ow and Lane (1988) Antibodies: A Labo rattro Manual, Cold Spring Harbor La laboratories, Cold Spring Harbor, NY and God ing (1986) Monoclonal Antibodies: Pri nchiples and Practice, Academic Press , By the standard procedure as described in New York City. Generated. Please refer to these two materials here. 11.Diagnostic test using HEDG-specific antibodies   Certain HEDG antibodies are directed to HEDG and downstream products of the activation signaling cascade. Diagnosis and signaling of infectious and genetic diseases characterized by differences in the amount or distribution of Useful for investigating transmission. HEDG was found in a specific human cDNA library Up-regulated mainly in immune and defense-related cell types Seems to have been.   Diagnostic testing methods for HEDG include body fluids, membranes, cells, tissues, Uses antibodies and labels to detect HEDG in extracts of such tissues How to include. The polypeptides and antibodies of the present invention may be used after modification, Or used without modification. The polypeptides and antibodies are often , Either covalent or non-covalent with a substance that transmits a detectable signal Labeled by binding with Various signs and related technologies are known. And has been widely reported in both scientific documents and patent specifications. Appropriate mark Knowledge includes radionuclides, enzymes, substrates, cofactors, inhibitors , Fluorescent agents, chemiluminescent agents, magnetic particles and the like. How to use such signs Are disclosed in U.S. Patent Application Nos. 3,817,837, 3,850,752, 39,350, 3,996,345, 4,277,437, 4,2 75,149, and 4,366,241. In addition Recombinant immunoglobulins can be obtained from those described in US Patent Application No. 4,816,567. Method. These specifications have been referenced together with this specification. No.   Polyclonal antibody or monoclonal specific for the protein Protease for the determination of soluble or membrane bound HEDG using any of the antibodies Various types of tokor are well known. An example of this is an enzyme-linked immuno Solvent assay (ELISA), radioimmunoassay (RIA), and fluorescence Activated cell sorting (FACS) is there. Preferred are those that react to two non-interfering epitopes on HEDG A two site monoclonal immunoassay using clonal antibodies However, a competitive binding assay may be used. These assays are, for example, Madd ox, DE, etc. (1983, J ExpMed 158: 1211) Other documents. 12.Purification of native HEDG using specific antibodies   Native or recombinant HEDG uses antibodies specific for HEDG. Purified by immuno-affinity chromatography. Generally, immune Affinity columns share anti-TRH antibody and activated chromatographic resin It is constituted by joining by joining.   Polyclonal immunoglobulin is precipitated with ammonium sulfate or Prepared from immune serum by purification on immobilized Protein A (Pha rmacia LKB Biotechnology, Piscataway NJ). Similarly, monoclonal antibodies can be prepared using ammonium sulfate precipitation or immobilized protein. It is prepared from mouse ascites by chromatography on protein A. Partial Immunoglobulin purified to CnBr-activated Sepharose (Pha chromatographs such as Rmcia LKB Biotechnology) Covalently bound to the resin. The antibody is bound to the resin by processing according to the manufacturer's instructions. The resin is blocked and the derivative resin is washed.   Such an immunoaffinity column prepares a fraction of cells containing HEDG This is used when purifying HEDG. This preparation should be performed on all cells To isolate cell fractions obtained using different centrifugation methods Or other known methods. Alternatively, signal distribution The soluble HEDG containing the sequence is secreted in amounts useful for the medium in which the cells grow.   The soluble HEDG containing the precipitate was passed through an immunoaffinity column, Conditions that allow preferential absorption of HEDG (ie, high ionic strength buffer in the presence of detergent) Is used). The column then cuts the antibody / protein binding. Conditions (ie, high concentrations of urea or thiocyanate ions). (Using an ototrope or a buffer of pH 2-3), and HEDG is recovered. Is done. 13.Drug screening   The present invention relates to HEDG or its binding flag in various drug screening techniques. It is particularly useful in the screening of therapeutic compounds using mentments. like this Polypeptides or fragments used in various tests are free to solutes Whether they are attached to the carrier of the individual, or are present on the cell surface or in the cell That's it. One method of drug screening involves expressing a propeptide or Eukaryote stably transformed with a recombinant nucleic acid or fragment thereof Use prokaryotic host cells. The drug is such in a competitive binding assay Is screened against the transformed cells. Such cells are Vesicles or fixed cells used for standard binding assays It is. For example, the formation of a complex between HEDG and the agonist to be tested is measured. In another example, the effect of the drug being tested on the complex formation between HEDG and the receptor Can also be tested.   Accordingly, the present invention provides a method for screening other drugs that affect signal transduction. It provides the law. The method comprises providing such a drug with a CALR polypeptide. Or (i) contacting the drug with the HEDG polypeptide. The presence of a complex with the peptide or fragment, or (ii) the HEDG polypeptide Assay for the presence of the complex of the cell or fragment with the cell using well-known methods. Including Such a competition In a binding assay, the HEDG polypeptide or fragment is usually Be labeled. After appropriate incubation, free HEDG polypeptide or Fragments are separated from those in bound form and may be free or uncomplexed. The amount of labeling depends on the ability of a particular drug to bind to HEDG, and the amount of HEDG and agonist. It is a measure of the ability to interfere with coalescence.   Other techniques for drug screening, suitable for HEDG polypeptides High throughput for screening compounds with high binding affinity This is described in European Patent No. 84, published on September 13, 1984. / 03564, to which reference is made here. simply To illustrate, a large number of different small peptide test compounds are Or synthesized on a solid substrate such as another surface. This peptide test compound After being reacted with the HEDG polypeptide, it is washed. HEDG PO combined The polypeptide is then detected using well-known methods. The purified HEDG is Coating directly on a plate for use in the drug screening techniques described above May be performed. In addition, the peptide is captured using a non-neutralizing antibody and It can also be immobilized on a carrier.   In the present invention, a neutralizing antibody capable of binding to HEDG is provided by a HEDG polypeptide or the same. Competitive drug screening assays that compete with test compounds that bind to fragments of The use of essays is also included. In this method, the antibody is bound to HEDG by one or more Used to detect the presence of any peptide sharing the above antigenic determinant. 14．Rational drug design   The purpose of rational drug design is to target the biologically active polypeptide or Are small molecules with which they interact, ie, agonists, To produce a gonist, or a structural analog of an inhibitor. these In any of the examples, the drug is more active and stable for the polypeptide, Or for use in forming drugs that interfere with polypeptide function in vivo Can be. (Hodgson J (1991) Bio / Technology 9: 19-21)   In one method, the protein of interest, or protein-inhibitor complex The three-dimensional structure of the body is determined by X-ray crystallography, computer modeling, or Type is determined by combining these two methods. Polypeptide Both the shape and the charge of Must be confirmed. Useful information about the structure of polypeptides It may be obtained by modeling based on the structure of the protein. Th In both cases, the resulting structural information can be used to design effective inhibitors. Used for Useful examples of rational drug design include Braxton San d Wells JA (1992 Biochemistry 31: 7766) A molecule with improved activity and stability as shown by -7801), or Athauda SB, etc. (1993 J Biochem 113: 742-7) Inhibitors, agonists, etc. of native peptides as shown by 46) Or molecules that function as antagonists. I want to be.   Isolating the target-specific antibody selected by the functional assay as described above, It is also possible to elucidate its crystal structure. This method, in principle, Form the pharmacore that is the basis for drug design. Functional and Produces anti-idiotypic antibodies (anti-ids) to pharmacologically active antibodies By doing so, it is also possible to skip the protein crystal analysis. Mirror image of mirror image In relation to, anti- The binding site of the ids is expected to be an analog of the original receptor. Then this anti-ids is a chemically or biologically generated peptide Ban Used to identify and isolate peptides from k. The isolated peptide is fur Functions as a macaco.   In accordance with the present invention, a sufficient amount of the polypeptide is formed, which is useful for X-ray crystallography. Can be used to perform such analytical studies. Further, the HED used here Knowing the G amino acid sequence allows it to be used instead of X-ray crystallography, or At the same time, guidance is provided for using computer modeling techniques. 15.Identification of other members of the signaling complex   Purified HEDG may be a G protein, or other signaling pathway protein. A research tool for identifying, characterizing, and purifying quality interactions. radiation Sex labels are incorporated into HEDG selected by various known methods and complement Used in vitro to capture molecules. The preferred method is HEDG In the primary amino group in¹²⁵I Bolton Hunter Reagent (Bolton, AE   and Hunter, WM (1973) Biochem J 133: 529). This reagent causes concomitant loss of biological activity It has been used to label various molecules without happening (Hebert C. A et al (1991) J Biol Chem 266: 18989. McColl S et al (1993) J Immunol 15; 0: 4550-4555).   Labeled HEDG is also useful as a reagent for purifying the molecules with which it interacts. It is for. In one embodiment of affinity purification, membrane-bound HEDG is chromatographed. Covalently bound to the chromatography column. Cell migration from putative target cells The extract is passed through the column and has the appropriate affinity. Binds to HEDG. HEDG complex is recovered from the column and Once released, the recovered molecule undergoes N-terminal protein sequencing. this Use the amino acid sequence to identify captured molecules or to use appropriate DNA libraries Modified oligonucleotide probe for cloning the target gene from Is designed.   In another method, antibodies to HEDG, especially monoclonal antibodies, are described above. Produced as This monoclonal antibody has been screened for And those that inhibit the binding of HEDG to HEDG are identified. Such monoclonal antibodies are used therapeutically. 16.Use and administration of HEDG antibodies, inhibitors or antagonists   HEDG antibodies, inhibitors, or antagonists (or other signals) (LST), a drug that limits transmission, exerts different effects when administered therapeutically I do. LST is a non-toxic, inert, pharmaceutically acceptable aqueous carrier medium. Are combined. The pH of the medium is preferably about 5 to 8, particularly preferably 6 to 8. However, this pH does not affect the characteristics of the incorporated antibody, inhibitor, or antagonist. Gender, and other conditions. LST properties include molecular solubility, halving A carrier in which these and other properties are effective Will help you decide. Although natural human proteins are also suitable as LSTs, Organic or synthetic molecules obtained from product screening may also be And it is equally effective.   LST is administered by well known routes of administration. This route of administration includes topical creams and Gels, transmucosal sprays and aerosols, transdermal patches and bandages, injectable veins Irrigation formulations and oral liquid and tablet drugs that are resistant to stomach acids and enzymes There are some that are formulated to have It is not limited to these. Hospital specific physician for specific formulation, correct dose, and route of administration , But may vary depending on the situation.   Such a determination depends on the treatment condition, the LST to be administered, and the pharmacodynamics of the particular LST. This is done by considering various factors, such as the biological profile. Consideration Other factors that should be included include the patient's condition (eg, whether it is severe), age, Weight, gender, diet, time, and frequency of administration, drug combinations, response sensitivities and treatment Resistance / reactivity. Long-acting LST formulation administration frequency Is administered once every 3 to 4 days, once a week, or once every two weeks However, this frequency is determined by the half-life and clearance rate of a particular LST. You.   Typical doses are between 0.1 and 100,000 μg, with an upper limit for total dose of about 1 g, depending on the route of administration. Specific dosages and methods of administration Guidance is provided in U.S. Patent Applications Nos. 4,657,760 and 5,206,344. Or, it is described in US Pat. No. 5,225,212. Those skilled in the art will be Use different formulations. For administration to cells such as nerve cells, vascular endothelial cells Different administration methods are required than for other cells such as vesicles.   In addition, conditions or diseases that can induce a disorder treatable by LST, Null transmission is conceivable. These conditions or diseases are diagnosed by the diagnostic tests described above. Such tests may include viral, bacterial or fungal infections, allergies Reaction, mechanical injury from trauma, genetic disease, lymphoma or carcinoma, or lymphatic tissue Should be performed when other conditions that activate the gene are suspected to have occurred .   All documents and patent specifications mentioned herein are incorporated herein by reference. I want to. Various modifications and alterations of the method and system of the invention described above are within the scope of the invention. And what can be done without departing from the spirit Will be obvious to others. Although a specific embodiment of the present invention has been described, It is not intended to limit the description to such specific embodiments. In fact, the present invention In implementation, various modifications of the above-described embodiments that will be apparent to those skilled in the art are set forth in the following claims. It is intended to be performed without departing from the scope of the invention as set forth.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C07K 16/28 C12N 1/19 C12N 1/19 1/21 1/21 C12P 21/02 C 5/10 G01N 33/53 D C12P 21/02 M G01N 33/53 33/566 C12P 21/08 33/566 C12N 5/00 B // C12P 21/08 A61K 37/02 (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, SZ, UG), UA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AT, AU, BR, CA, CH, CN, E, DK, ES, FI, GB, IL, JP, KR, MX, NO, NZ, RU, SE, SG (72) Inventor Oo-Young, Janice Berkeley Kinds Avenue, CA 94702, California 1419 (72) Inventor Bandman, Olga 94043, Mountain View Rock Street, California, U.S.A. # 27 2309 (72) Inventor Sealheimer, Jeffrey J. Los Altos Hills Lacresta, California, U.S.A.

Claims (1)

[Claims] 1. A nucleic acid sequence encoding the polypeptide of SEQ ID NO: 2 or its complementary sequence A purified polynucleotide comprising. 2. 2. The polynucleotide of claim 1, comprising the nucleic acid sequence (hedg) of SEQ ID NO: 1. Chid. 3. An amplifier comprising the complementary sequence of the polynucleotide of claim 2 or a portion thereof. Chisense molecule. 4. A pharmaceutical set comprising the antisense molecule of claim 3 and a pharmaceutically acceptable excipient. Adult. 5. Administering to the patient an effective amount of the pharmaceutical composition of claim 4. A method for treating a patient suffering from a condition associated with abnormal expression of hedg, characterized by the following. 6. A diagnostic composition comprising an oligomer of the polynucleotide of claim 2. 7. A diagnostic test method for a condition associated with abnormal expression of hedg,   a) preparing a biological sample;   b) combining the biological sample with the pharmaceutical composition of claim 6;   c) under appropriate conditions, hybridizing the biological sample with the diagnostic composition. The process of forming a state where the formation of   d) measuring the level of hybridization to obtain a sample value;   e) comparing the value of the sample with a standard value to determine whether the hedg expression is abnormal; Determining the condition associated with the abnormal expression of hedg. Diagnostic test method. 8. An expression vector comprising the polynucleotide of claim 1. 9. A host cell transformed with the expression vector according to claim 8. 10. A method for producing a polypeptide, comprising:   a) culturing the host cell of claim 9 under conditions suitable for expression of said polypeptide. Process,   b) recovering the polypeptide from the host cell medium. A method for producing a characteristic polypeptide. 11. A resulting polypeptide comprising the amino acid sequence of SEQ ID NO: 2 (HEDG). 12. A diagnostic composition comprising the polypeptide of claim 11 or a portion thereof. 13. A pharmaceutical set comprising the polypeptide of claim 11 and a pharmaceutically acceptable excipient. Adult. 14． Administering to the patient an effective amount of the pharmaceutical composition of claim 13. A method for treating a patient suffering from a condition associated with abnormal expression of HEDG. 15. An antibody specific for the purified polypeptide of claim 11 or a portion thereof. 16. A diagnostic composition comprising the antibody of claim 15. 17． A diagnostic test method for a condition associated with abnormal expression of HEDG,   a) preparing a biological sample;   b) combining the biological sample with the antibody of claim 15 under conditions suitable for complex formation. Combining the   c) measuring the level of complex formation between HEDG and the antibody and determining the level value of the sample; The process of getting   d) comparing the level of complex formation in the sample with the standard value of complex formation. And the process of   The deviation of the level value of the sample from the standard level value of the complex formation Diagnosis of a condition associated with abnormal expression of HEDG, characterized in that its presence is confirmed Strike method. 18. An affinity that specifically binds to the polypeptide of claim 11 or any portion thereof. A method of screening a plurality of compounds by gender,   a) preparing a plurality of compounds;   b) Under sufficient conditions, take sufficient time for binding to take place, And binding each of the plurality of compounds;   c) detecting the binding of each of the plurality of compounds to HEDG, Identifying a compound that binds differentially. Multipleization by affinity specifically binding to the polypeptide or any part thereof A method for screening compounds. 19. A pharmaceutical composition comprising the compound of claim 18 and a pharmaceutically acceptable excipient. . 20. 20. A method comprising administering to the patient an effective amount of the pharmaceutical composition of claim 19. A method for treating a patient suffering from a condition associated with abnormal expression of EDG.