JP2002503635A - EPRG3Spt, EPO primary response gene - Google Patents

Abstract

  (57) [Summary]   EPRG3Spt polypeptides and polynucleotides and methods of producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods of using EPRG3Spt polypeptides and polynucleotides for therapy, and diagnostic assays for such uses.

Description

DETAILED DESCRIPTION OF THE INVENTION

This application claims the benefit of US Provisional Application No. 60 / 054,464 (filed August 1, 1997), the disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION [0002] The present invention relates to newly identified polypeptides, polynucleotides encoding the polypeptides, and agonists that may be effective in or for the treatment of the polypeptides and polynucleotides. , Antagonists and / or inhibitors, and methods of producing such polypeptides and polynucleotides.

[0003] The drug discovery process is currently undergoing a radical change. This is because it extends to "functional genomics", that is, high-throughput (high-efficiency) genomic or gene-based biology. This approach is rapidly replacing a relatively early approach based on “positional cloning”. The phenotype, ie, biological function or genetic disease, will be identified and the etiological gene will then be located based on its genetic map location.

[0004] Functional genetic science relies heavily on various bioinformatics tools to identify gene sequences of interest from many currently available molecular biology databases. There still exists a need to identify and characterize yet-unknown genes and their related polypeptides / proteins as targets for drug discovery.

SUMMARY OF THE INVENTION [0005] The present invention relates to EPRG3Spt, particularly EPRG3Spt polypeptides and polynucleotides, recombinant materials, and methods for producing the same. In another embodiment, the present invention relates to the aforementioned polypeptides and polynucleotides, including the treatment of anemia, erythrocytosis, cancer, AIDS, and drug-induced anemia (hereinafter collectively referred to as “the disease”). How to use. In other aspects, the present invention relates to methods of identifying agonists and antagonists / inhibitors using the agents provided by the invention, and to treating conditions associated with EPRG3Spt imbalance using the identified compounds. In yet another embodiment, the present invention provides a method for treating an inappropriate EPRG3Spt
Diagnostic assays for detecting diseases associated with activity or EPRG3Spt levels.

Description of the Invention In one embodiment, the invention is directed to an EPRG3Spt polypeptide. This kind of peptide has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2. Comprises an isolated polypeptide comprising an amino acid sequence having at least 95% identity, most preferably at least 97-99% identity. Such polypeptides include polypeptides comprising the amino acid sequence of SEQ ID NO: 2.

[0007] Other peptides of the invention have at least 70% identity, preferably at least 80% identity, more preferably at least 70% identity to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2 Isolated polypeptides having at least 90% identity, more preferably at least 95% identity, and most preferably at least 97-99% identity are included. Such polypeptides include polypeptides consisting of the amino acid sequence of SEQ ID NO: 2.

[0008] Further peptides of the invention include an isolated polypeptide encoded by a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 1.

[0009] The polypeptides of the present invention are human p27 (Rasmussen, UB et al., 1993, Cancer Research).
53: 4096-4101) and may be structurally related to other proteins that have homology and / or structural similarity to the protein. Therefore, they are interesting. Because the expression of certain proteins is induced by EPO, and the presence of EPO is required to maintain the expression of such proteins. These EPO-related proteins are EPO
It is involved in the growth of dependent cells and may be important in the growth and development of erythroid and other hematopoietic lineages. These properties will be referred to as “EPRG3Spt activity” or “EPRG
It is referred to as "RG3Spt polypeptide activity" or "biological activity of EPRG3Spt". These activities include the antigenic activity and the immunological activity of the EPRG3Spt polypeptide, particularly the antigenic activity and the immunological activity of the polypeptide of SEQ ID NO: 2. Preferably, the polypeptide of the present invention exhibits at least one biological activity of EPRG3Spt.

[0010] The polypeptides of the present invention may be in the form of the "mature" protein or may be part of a larger protein, such as a fusion protein. It is often advantageous to include additional amino acid sequences, such as secretory or leader sequences, prosequences, sequences useful for purification such as multiple histidine residues, or stable during recombinant production. There are additional arrangements to ensure the performance.

[0011] The present invention also includes a mutant of the above-mentioned polypeptide, that is, a polypeptide which differs from a reference polypeptide by a conservative amino acid substitution (a certain residue is substituted by another residue having similar properties). include. Typical such substitutions are Ala, Val, Le
between u and Ile; between Ser and Thr; between the acidic residues Asp and Glu; between Asn and Gln; between the basic residues Lys and Arg; or between the aromatic residues Phe and Tyr. In particular, a mutant in which several, 5 to 10, 1 to 5, 1 to 3, 1 to 2, or 1 amino acids are substituted, deleted, or added in any combination is preferable.

[0012] The polypeptide of the present invention can be produced by any appropriate method. Such polypeptides include isolated natural polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. It is. The means for producing such polypeptides are well understood in the art.

[0013] In a further aspect, the present invention relates to an EPRG3Spt polynucleotide. Such a polynucleotide has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2, Preferably, an isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having at least 95% identity is included. At least 97% in this regard
Are more preferred, those with at least 98-99% identity are even more preferred, and those with at least 99% identity are most preferred. Such polynucleotides include polynucleotides comprising the nucleotide sequence contained in SEQ ID NO: 1 encoding the polypeptide of SEQ ID NO: 2.

[0014] Further polynucleotides of the present invention include at least 70% of the nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 over its entire coding region.
Identity, preferably at least 80% identity, more preferably at least 9%
An isolated polynucleotide comprising a nucleotide sequence having 0% identity, more preferably at least 95% identity, is included. In this regard, polynucleotides having at least 97% identity are more preferred, while those with at least 98-99% identity are even more preferred, and those with at least 99% identity are most preferred. .

[0015] Additional polynucleotides of the present invention include SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.
A nucleotide sequence having at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably at least 95% identity to a polynucleotide of Isolated polynucleotides are included. In this regard, polynucleotides having at least 97% identity are more preferred, while those with at least 98-99% identity are even more preferred, and those with at least 99% identity are most preferred. . Such polynucleotides include a polynucleotide comprising the polynucleotide of SEQ ID NO: 1 and a polynucleotide of SEQ ID NO: 1.

The present invention also provides polynucleotides that are complementary to all of the above polynucleotides.

The nucleotide sequence of SEQ ID NO: 1 is human p27 (Rasmussen, UB et al., 1993, Cancer R
esearch 53: 4096-4101). The nucleotide sequence of SEQ ID NO: 1 is a cDNA sequence, and a polypeptide coding sequence encoding a polypeptide consisting of 100 amino acids which is the polypeptide of SEQ ID NO: 2 (nucleotide numbers 595 to 897)
including. Even though the nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 is identical to the polypeptide coding sequence contained in SEQ ID NO: 1, the polypeptide of SEQ ID NO: 2 is still duplicated due to the redundancy (degeneracy) of the genetic code. It may be a sequence other than the sequence contained in SEQ ID NO: 1 that encodes. The polypeptide of SEQ ID NO: 2 is human p27 (
Rasmussen, UB et al., 1993, Cancer Research 53: 4096-4101) and are structurally related to other proteins having homology and / or structural similarity.

Preferred polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions / properties as the polypeptides and polynucleotides homologous thereto. Further, preferred polypeptides and polynucleotides of the present invention have at least one EPRG3Spt activity.

The present invention also relates to partial or other polynucleotides and polypeptides initially identified prior to the determination of the corresponding full length sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

Thus, in a further aspect, the invention provides (a) at least 70% identity, preferably at least 80% identity, more preferably at least 80% identity to the nucleotide sequence of SEQ ID NO: 3 over the entire length of SEQ ID NO: 3 Is a nucleotide sequence having at least 90% identity, more preferably at least 95% identity, most preferably 97-99% identity; (b) relative to the nucleotide sequence of SEQ ID NO: 3 over the entire length of SEQ ID NO: 3 Nucleotides having at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably at least 95% identity, and most preferably 97-99% identity. Or an isolated polynucleotide comprising (c) a polynucleotide of SEQ ID NO: 3. To provide.

Thus, in a further aspect, the present invention provides: (a) at least 70% identity, preferably at least 80% identity, more preferably at least 80% identity to the nucleotide sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4 A nucleotide sequence having at least 90% identity, more preferably at least 95% identity, most preferably 97-99% identity; (b) relative to the nucleotide sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4 A nucleotide sequence having at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably at least 95% identity, and most preferably 97-99% identity. Or (c) a polynucleotide of SEQ ID NO: 4, an isolated polynucleotide comprising To provide.

The nucleotide sequence of SEQ ID NOs: 3 and 4 and the peptide sequence encoded thereby are expressed in an Expressed Sequence Tag (EST).
) Derived from the sequence. One skilled in the art will appreciate that there will necessarily be some nucleotide sequence reading errors in the EST sequence (Adams, MD et al., N.
ature 377 (supp) 3, 1995). Accordingly, the nucleotide sequences of SEQ ID NOs: 3 and 4 and the peptide sequences encoded thereby suffer from the same inherent limitations in sequence accuracy. In addition, the peptide sequences encoded by SEQ ID NOs: 3 and 4 may be of the same region as the protein with the highest homology or structural similarity, or of high homology and / or structural similarity (eg, conservative amino acid differences). Contains the area.

[0023] The polynucleotides of the present invention may comprise cDNA derived from mRNA in human bone marrow and hematopoietic cells by standard cloning and screening techniques.
From the library, EST analysis (Adams, MD et al., Science (1991) 252: 1651-165)
6; Adams, MD et al., Nature (1992) 355: 632-634; Adams, MD et al., Nature (1995
377 Supp: 3-174). In addition, the polynucleotide of the present invention can be obtained from a natural source such as a genomic DNA library, and can also be synthesized using a commercially available known technique.

When a polynucleotide of the present invention is used for recombinant production of a polypeptide of the present invention, the polynucleotide may include the coding sequence for the mature polypeptide alone, or another coding sequence (eg, a leader or secretory sequence). Sequences, those encoding the pre-, pro- or pre-pro-protein sequences, or other fusion peptide portions), and the coding sequence of the mature polypeptide in the same reading frame. For example, a marker sequence that facilitates purification of the fusion polypeptide can be encoded. In a preferred embodiment of this aspect of the invention, the marker sequence is a pQE vector
(Qiagen, Inc.) and Gentz et al., Proc. Natl. Acad. Sci. USA (19
89) Hexa-histidine peptide or HA tag as described in 86: 821-824. Also, the polynucleotide may have 5 'and 3' non-coding sequences, e.g.,
It may include transcribed but not translated sequences, splicing and polyadenylation signals, ribosome binding sites, and mRNA stabilizing sequences.

Further specific examples of the present invention include several, for example, 5 to 10, 1 to 5, 1 to 3,
There is a polynucleotide encoding a polypeptide variant comprising the amino acid sequence of SEQ ID NO: 2, wherein one, two, or one amino acid residue is substituted, deleted or added in any combination. .

A polynucleotide that is identical or sufficiently identical to the nucleotide sequence contained in SEQ ID NO: 1 can be used to isolate full length cDNA and genomic clones encoding a polypeptide of the present invention. To isolate cDNA and genomic clones of other genes with high sequence similarity to, including homologues from non-human species and genes encoding orthologs,
It can be used as a hybridization probe for DNA and genomic DNA, or as a primer for a nucleic acid amplification (PCR) reaction. Typically,
These nucleotide sequences are 70%, preferably 80%, of the reference nucleotide sequence.
%, More preferably 90%, and most preferably 95%. Probes or primers usually contain 15 or more nucleotides, preferably 30 or more, and may have 50 or more nucleotides. Particularly preferred probes are those having a range of 30 to 50 nucleotides.

Polynucleotides encoding the polypeptides of the present invention (including homologs and orthologs from non-human species) can be stringent using labeled probes having the sequence of SEQ ID NO: 1 or fragments thereof. It is obtained by a method comprising the steps of screening an appropriate library under hybridization conditions and isolating a full-length cDNA and a genomic clone containing the polynucleotide sequence. Such hybridization techniques are known to those skilled in the art. Preferred stringent hybridization conditions are 50% formamide, 5 × SSC
(150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),
Incubate overnight at 42 ° C. in a solution containing 5 × Denhardt solution, 10% dextran sulfate and 20 μg / ml denatured sheared salmon sperm DNA, then wash the filters at about 65 ° C. in 0.1 × SSC. Thus, the present invention also includes a polynucleotide obtained by screening an appropriate library under stringent hybridization conditions using a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof.

As will be appreciated by those skilled in the art, the isolated cDNA sequence will often be incomplete, since in many cases the region encoding the polypeptide is truncated at the 5 ′ end of its cDNA. Would. It is due to reverse transcriptase, which originally has a low "processivity" (a measure of the enzyme's ability to remain attached to the template during the polymerization reaction), and the mRNA template during first strand cDNA synthesis. DNA copy cannot be completed.

There are several methods known and available to those skilled in the art for obtaining full-length cDNAs or elongating short-chain cDNAs, such as the cDNA end rapid amplification method (R
ACE) (see, eg, Frohman et al., PNAS USA 85, 8998-9002,
1988). Recent improvements in the above techniques, as demonstrated, for example, by Marathon ^™ technology (Clontech Laboratories Inc.), have greatly simplified the search for longer cDNAs. With Marathon ^™ technology, mRNA extracted from a given tissue
And an "adapter" sequence is ligated to each end. Subsequently, nucleic acid amplification (PCR) is performed using a combination of gene-specific and adapter-specific oligonucleotide primers to amplify the "deleted" 5 'end of the cDNA. next,
"Nested" primers, ie, primers designed to anneal inside the amplification product (typically an adapter-specific primer that anneals further 3 'to the adapter sequence and an additional 5' to the known gene sequence The PCR reaction is repeated using the gene-specific primers. The product of this reaction is then analyzed by DNA sequencing and the product is directly linked to the existing cDNA to complete the sequence, or another full-length sequence using new sequence information for 5 'primer design. By performing PCR, a full-length cDNA can be constructed.

[0030] The recombinant polypeptides of the present invention can be produced from genetically engineered host cells containing the expression system using methods known in the art. Thus, in a further aspect, the present invention relates to an expression system containing one or more polynucleotides of the present invention, a host cell engineered with said expression system, and the production of a polypeptide of the present invention by recombinant methods. Cell-free translation systems can also be used to produce such proteins using RNA derived from the DNA constructs of the present invention.

For recombinant production, the host cell is genetically engineered to incorporate the polynucleotide expression system of the invention or a portion thereof. Introduction of polynucleotides into host cells is described in Davis et al., Basic Methods in Molecular Biology (1986) and Sambro.
ok et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbo
r Laboratory Press, Cold Spring Harbor, NY (1989) and many other standard laboratory manuals. Suitable such methods include, for example, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape loading, Bullet introduction (b
allistic introduction) or infection.

Representative examples of suitable hosts include bacterial cells (eg, Streptococcus, Staphylococcus, E. coli, Streptomyces, Bacillus subtilis), fungal cells (eg, yeast, Aspergillus), insect cells (eg, Drosophila S2) , Spodoptera Sf9), animal cells (eg, CHO, COS, HeLa, C127, 3T3, BHK, HEK293)
, Bowes melanoma cells) and plant cells.

[0033] A wide variety of expression systems can be used. Such expression systems include, for example,
Systems derived from chromosomes, episomes and viruses, such as bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion factors, yeast chromosomal elements, viruses (eg, baculovirus, papovavirus such as SV40, vaccinia virus , Adenovirus, fowlpox virus,
Pseudorabies virus, retrovirus) and vectors derived from combinations thereof, for example, those derived from plasmid and bacteriophage genetic elements such as cosmids and phagemids. These expression systems may contain control regions that regulate as well as cause expression. Typically,
Any system or vector capable of maintaining, augmenting, and expressing a polynucleotide for production of a polypeptide in a host may be used. Sambrook et al., Molecu
The appropriate nucleotide sequence can be inserted into the expression system by any of the commonly used and known techniques, such as those described in lar Cloning: A Laboratory Manual (supra). Appropriate secretion signals can be incorporated into the polypeptide of interest to secrete the translated protein into the endoplasmic reticulum lumen, into the periplasmic space, or into the extracellular environment. These signals may be endogenous to the polypeptide of interest or heterologous signals.

When it is desired to express a polypeptide of the present invention for use in a screening assay, it is generally preferred that the polypeptide be produced on the surface of cells. If so, the cells are harvested prior to use in the screening assay.
If the polypeptide is secreted into the medium, the medium is recovered to recover and purify the polypeptide. If produced intracellularly, it is necessary to first lyse the cell and then recover the polypeptide.

To recover and purify the polypeptide of the present invention from recombinant cell culture, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity Known methods including chromatography, hydroxylapatite chromatography, and lectin chromatography can be used. Most preferably, high performance liquid chromatography is used for purification. When the polypeptide is denatured during intracellular synthesis, isolation and / or purification, it is possible to restore the active conformation using known techniques for regenerating the protein.

The present invention also relates to the use of the polynucleotide of the present invention as a diagnostic. Detection of a variant of a gene characterized by a polynucleotide of SEQ ID NO: 1 associated with a dysfunction is useful for diagnosing a disease caused by under-expression, over-expression or altered expression of the gene or susceptibility to the disease. It will provide a diagnostic tool that can be added or diagnosed. Individuals with mutations in the gene can be found at the DNA level by various techniques.

Diagnostic nucleic acids can be obtained from cells of a subject, such as cells from blood, urine, saliva, tissue biopsy or autopsy material. Genomic DNA may be used directly for detection, or may be enzymatically amplified using PCR or other amplification methods prior to analysis. RNA or cDNA can be used in a similar manner. Deletions and insertions can be detected by a change in the size of the amplified product when compared to the normal genotype. Point mutations can be identified by hybridizing the amplified DNA to a labeled EPRG3Spt nucleotide sequence. Perfectly matched sequences and mismatched duplexes can be distinguished by RNase digestion or by differences in melting temperatures. DNA sequence differences can also be detected by changes in the electrophoretic mobility of the DNA fragments on gels with or without denaturing agents, or by direct DNA sequencing (eg, Myers et al., Science (1985)). 230: 1242). Sequence changes at specific positions can also be confirmed by nuclease protection assays (eg, RNase and S1 protection) or by chemical cleavage (Cotton et al.).
Natl. Acad. Sci. USA (1985) 85: 4397-4401). In another embodiment, for example, to perform efficient screening for gene mutations, EPRG3S
Array of oligonucleotide probes containing pt nucleotide sequences or fragments thereof
(array) can be constructed. Array techniques are known and have general applicability and are used to solve a variety of molecular genetics problems, including gene expression, genetic linkage and genetic variability (eg, M Chee et al., Science, Vo
l.274, pp.610-613 (1996)).

The diagnostic assay comprises detecting a mutation in the EPRG3Spt gene according to the method described above,
A method is provided for diagnosing or determining susceptibility to the disease. In addition, the disease can be diagnosed by a method of measuring an abnormal decrease or increase in the level of polypeptide or mRNA from a sample obtained from a subject. Decreased or increased expression can be determined by methods known in the art for quantifying polynucleotides, such as nucleic acid amplification (
Examples: PCR, RT-PCR), RNase protection, Northern blotting, and other hybridization methods. Assays that can be used to determine levels of a protein, such as a polypeptide of the present invention, in a sample obtained from a host are well-known to those of skill in the art. Such assays include radioimmunoassays, competitive binding assays, western blot analysis, ELISA assays and the like.

Thus, in another embodiment, the present invention provides: (a) a polynucleotide of the present invention (preferably the nucleotide sequence of SEQ ID NO: 1) or a fragment thereof, (b) complementary to the nucleotide sequence of (a). (C) an antibody against the polypeptide of the present invention (preferably, the polypeptide of SEQ ID NO: 2) or a fragment thereof, or (d) an antibody against the polypeptide of the present invention (preferably, the polypeptide of SEQ ID NO: 2), And a diagnostic kit comprising:

It will be appreciated that in such a kit, (a), (b), (c) or (d) is a substantial component. Such kits are useful in diagnosing a disease or susceptibility to a disease, particularly anemia, polycythemia, cancer, AIDS, and drug-induced anemia.

[0041] The nucleotide sequence of the present invention is also useful for chromosome identification. This sequence can specifically target a particular location on an individual human chromosome and hybridize to that particular location. Creating a chromosomal map of related sequences according to the present invention is an important first step in correlating these sequences with gene-related diseases. Once a sequence has been mapped to a precise chromosomal location, the physical location of that sequence on that chromosome can be correlated with genetic map data. This type of data is described, for example, in V. McKusick, Mendelian Inheritance in Man (Johns Hopki
(available online at ns University Welch Medical Library). Thereafter, the relationship between the gene mapped to the same chromosomal region and the disease is confirmed by linkage analysis (co-inheritance of physically adjacent genes).

[0042] Differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation may be responsible for the disease.

The gene of the present invention maps to human chromosome 14q31-q32. Disorders at this locus have been identified in some lymphoid malignancies.

The polypeptide of the present invention, a fragment or an analog thereof, or a cell expressing the same can also be used as an immunogen for producing an antibody immunospecific for the polypeptide of the present invention. By "immunospecific" is meant that the antibody exhibits a substantially higher affinity for the polypeptides of the present invention than its affinity for other related polypeptides in the prior art.

[0045] Antibodies to the polypeptides of the present invention can be prepared in animals (using conventional protocols).
(Preferably a non-human animal) by administering a fragment, analog or cell containing the polypeptide or epitope. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. For example, the hybridoma method (Kohler, G. and Milstein, C., Nature (
1975) 256: 495-497), trioma method, human B cell hybridoma method (Kozbor et al.,
Immunology Today (1983) 4:72) and the EBV-hybridoma method (Cole et al., Mon.
oclonal Antibodies and Cancer Therapy, pp.77-96, Alan R. Liss, Inc., 198
5) and so on.

To generate single chain antibodies to a polypeptide of the invention, US Pat. No. 4,946,
Methods for preparing single chain antibodies as described in US Pat. No. 778 can be adapted. Also, transgenic mice or other organisms, including other mammals, can be used to express humanized antibodies.

Using the antibody described above, a clone expressing the polypeptide can be isolated and identified, or the polypeptide can be purified by affinity chromatography.

[0048] Antibodies to the polypeptides of the present invention may be used, inter alia, for the treatment of the aforementioned diseases.

In a further aspect of the present invention, the present invention provides a polypeptide of the present invention or a fragment thereof and a variety of H or L chain constant regions of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE). And a genetically engineered soluble fusion protein comprising: The immunoglobulin is preferably the constant part of the heavy chain of human IgG, especially IgG1, in which case the fusion takes place in the hinge region. In certain instances, the Fc portion can be easily removed by incorporating a cleavage sequence that can be cleaved by blood coagulation factor Xa. Furthermore, the invention relates to methods for the genetic engineering of these fusion proteins and their use in drug screening, diagnosis and therapy. A further aspect of the present invention relates to a polynucleotide encoding such a fusion protein. Examples of fusion protein technology are described in International Patent Application WO94 / 2945
8 and WO94 / 22914.

A further aspect of the invention relates to a method of eliciting an immunological response in a mammal,
The method comprises inoculating a mammal with an antibody and / or a polypeptide of the invention sufficient to generate a T cell immune response, particularly to protect the animal from the disease. Yet another aspect of the invention directs the expression of a polynucleotide encoding a polypeptide of the invention in vivo to elicit an immunological response that produces antibodies that protect the mammal from the disease. A method of eliciting an immunological response in a mammal, comprising providing the polypeptide via a vector.

A further aspect of the present invention relates to immunological / vaccine formulations (compositions) that, when introduced into a mammalian host, elicit an immunological response against the polypeptide of the present invention in the mammal. Contains a polypeptide or polynucleotide of the invention. The vaccine formulation may further comprise a suitable carrier. Since the polypeptide may be broken down in the stomach, it is preferably administered parenterally (eg, by subcutaneous, intramuscular, intravenous or intradermal injection). Formulations suitable for parenteral administration include sterile aqueous and non-aqueous injections, which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient, and suspensions. There are aqueous and non-aqueous sterile suspensions which may contain agents or thickeners. Such formulations may be presented in single or multi-dose containers (eg, sealed ampules and vials) and may also be stored in a lyophilized condition which requires only the addition of a sterile liquid carrier immediately before use. it can. The vaccine formulation may include an adjuvant system to enhance the immunogenicity of the formulation, such as an oil-in-water adjuvant system or other adjuvant systems known in the art. The dose varies with the specific activity of the vaccine, but can be easily determined by routine experimentation.

[0052] The polypeptides of the present invention are involved in a variety of biological functions, including a number of pathological conditions, particularly the aforementioned diseases. Therefore, it is desirable to develop screening methods that identify compounds that stimulate or suppress the function of this polypeptide. Thus, in a further aspect, the present invention provides a method of screening compounds to identify compounds that stimulate or suppress the polypeptide. In general,
Agonists or antagonists are used for the purpose of treating and preventing the above diseases. Compounds can be identified from a variety of sources, such as mixtures of cells, cell-free preparations, chemical libraries and natural products. The agonist, antagonist or inhibitor so identified may optionally be a natural or modified substrate, ligand, receptor, enzyme, etc., of the polypeptide, and its structural or functional mimetic. (Coligan et al., C
urrent Protocols in Immunology 1 (2): Chapter 5 (1991)).

In the screening method, binding of the candidate compound to the polypeptide of the present invention, or a cell or membrane carrying the polypeptide, or a fusion protein thereof is performed using a label directly or indirectly bound to the candidate compound. And easy to measure. Alternatively, the screening method may use competition with a labeled competitor.
Furthermore, such screening methods can test whether a candidate compound results in a signal resulting from activation or suppression of the polypeptide, using a detection system suitable for cells carrying the polypeptide. . Generally, inhibitors of activation are assayed in the presence of a known agonist to determine the effect of the presence of the candidate compound on activation by the agonist. Constitutively active polypeptides are used in screening methods for inverse agonists or inhibitors by examining whether a candidate compound inhibits activation of a polypeptide in the absence of an agonist or inhibitor. In addition, these screening methods
It simply involves mixing the candidate compound with a solution containing the polypeptide of the invention to form a mixture, measuring the EPRG3Spt activity in the mixture, and comparing the EPRG3Spt activity of the mixture to a standard. In high-throughput screening assays to identify antagonists of the polypeptides of the invention, fusion proteins such as those made from the Fc portion and the EPRG3Spt polypeptides described above can also be used (D. Bennett et al., J. Mol. Recognition, 8: 52-58 (199
5) and K. Johanson et al., J. Biol. Chem., 270 (16): 9459-9471 (1995)).

In addition, using the polynucleotide, polypeptide or antibody against the polypeptide of the present invention, a screening method for detecting the effect of an additional compound on the production of mRNA or polypeptide in cells may be assembled. it can. For example, using a monoclonal or polyclonal antibody by standard methods known in the art to determine the level of polypeptide secretion or cell binding
ISA assays can be constructed, which can be used to search for substances (also referred to as antagonists or agonists, respectively) that suppress or enhance the production of polypeptides from appropriately engineered cells or tissues.

If a membrane-bound or soluble receptor is present, the polypeptide of the invention may be used to identify such receptor by standard receptor binding methods known in the art. Can be used. Such receptor binding methods include, but are not limited to, ligand binding assays and cross-linking assays, in which the polypeptide is labeled with a radioactive isotope (eg, ¹²⁵ I) or chemically modified (eg, ¹²⁵ I). Eg, biotinylated) or fused to a peptide sequence suitable for detection and purification and incubated with a putative receptor source (cells, cell membranes, cell supernatants, tissue extracts, body fluids, etc.). Other methods include biophysical methods such as surface plasmon resonance and spectroscopy. These screening methods can also be used to identify agonists or antagonists of the polypeptide that compete for binding to the polypeptide or its receptor, if present. Standard methods for performing screening assays are well understood in the art.

Examples of potential antagonists of the polypeptides of the present invention include antibodies, and in some cases, oligonucleotides or proteins (eg, oligonucleotides or proteins) that are closely related to the ligands, substrates, receptors, enzymes, etc., of the polypeptide. , Ligands, substrates, receptors,
Fragments, such as enzymes) or small molecules that bind to a polypeptide of the invention but do not elicit a response (and thus prevent the activity of the polypeptide).

Thus, in other aspects, the present invention identifies agonists, antagonists, ligands, receptors, substrates, enzymes, etc., of the polypeptides of the invention, or compounds that decrease or increase the production of such polypeptides. This kit comprises: (a) a polypeptide of the present invention, (b) a recombinant cell expressing the polypeptide of the present invention, and (c) expressing the polypeptide of the present invention. A cell membrane, or (d) an antibody against the polypeptide of the present invention, wherein said polypeptide is preferably the polypeptide of SEQ ID NO: 2. It will be appreciated that in such a kit, (a), (b), (c) or (d) is a substantial component.

Those of skill in the art will readily appreciate that polypeptides of the present invention can also be used in methods for designing an agonist, antagonist or inhibitor of the polypeptide based on its structure. This method comprises the steps of (a) first analyzing the three-dimensional structure of the polypeptide, (b) assuming the three-dimensional structure of a likely reactive or binding site of an agonist, antagonist or inhibitor; Synthesizing a candidate compound that is expected to bind or react with the selected reactive or binding site, and (d) determining whether the candidate compound is in fact an agonist, antagonist or inhibitor. It will be further appreciated that this is usually an interactive process.

In a further embodiment, the present invention relates to abnormalities related to either an excess or deficiency of EPRG3Spt polypeptide activity, such as anemia, erythrocytosis, cancer, AIDS, and drug-induced anemia. To provide a cure for any condition.

If the activity of the polypeptide is in excess, several approaches are available. One approach is to suppress the function of the polypeptide, for example, by blocking the binding of ligands, substrates, receptors, enzymes, etc., or by reducing an abnormal condition by suppressing a second signal. Administering an inhibitor compound (antagonist) as described above to a patient in an amount effective to do so with a pharmaceutically acceptable carrier. In another approach, soluble forms of the polypeptide that are still capable of binding ligands, substrates, enzymes, receptors, etc., in competition with the endogenous polypeptide can be administered. A typical example of such a competitor is a fragment of the EPRG3Spt polypeptide.

In yet another approach, expression of the gene encoding endogenous EPRG3Spt polypeptide can be suppressed using expression blocking techniques. Such known techniques require the use of antisense sequences produced in the body or administered separately (eg, Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (oligodeoxynucleotides as antisense inhibitors of gene expression), CRC
Press, Boca Raton, FL (1988), O'Connor, J. Neurochem (1991) 56: 560). Alternatively, an oligonucleotide that forms a triple helix with this gene can be provided (see, for example, Lee et al., Nucleic Acids Res (1979).
6: 3073; Cooney et al., Science (1988) 241: 456; Dervan et al., Science (1991) 251.
: 1360). These oligomers can be administered as such, or the relevant oligomer can be expressed in vivo.

For treating abnormal conditions related to an under-expression of EPRG3Spt and its activity,
Several approaches can be taken. One approach involves administering to a patient a therapeutically effective amount of a compound that activates a polypeptide of the invention (ie, the agonist) together with a pharmaceutically acceptable carrier to alleviate the abnormal condition. It becomes. Alternatively, gene therapy can be used to produce EPRG3Spt endogenously in the relevant cells of the patient. For example, a polynucleotide of the invention may be engineered for expression by a replication defective retroviral vector as described above. The retroviral expression construct is then isolated and introduced into packaging cells transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention. As a result, the packaging cells will produce infectious viral particles containing the gene of interest. In vivo cell manipulation and
These producer cells are administered to a patient for in vivo polypeptide expression. For an overview of gene therapy, see Human Molecular Genetics, T Strachana.
Chapter 20, Gene T in nd AP Read, BIOS Scientific Publishers Ltd (1996)
See herapy and other Molecular Genetic-based Therapeutic Approaches (and references therein). Another approach is to administer a therapeutic amount of a polypeptide of the invention together with a suitable pharmaceutical carrier.

In a further aspect, the invention provides a therapeutically effective amount of a polypeptide (eg, a soluble form of the polypeptide of the invention), an agonist or antagonist peptide, or a small molecule compound in a pharmaceutically acceptable carrier or Pharmaceutical compositions are provided that contain together with excipients. Such carriers include, but are not limited to, saline, saline, dextrose, water, glycerol, ethanol, and combinations thereof. The present invention further provides a composition comprising one or more of the above-described compositions of the present invention.
It relates to a pharmaceutical pack and a kit comprising the above container. The polypeptides and other compounds of the present invention may be used alone or in conjunction with other compounds, eg, therapeutic compounds.

The pharmaceutical composition can be adapted to the route of administration, for example by a systemic or an oral route. A suitable form for systemic administration is infusion (injection), typically intravenous.
Other injection routes, such as subcutaneous, intramuscular or intraperitoneal, can also be used. Alternative means of systemic administration include transmucosal and transdermal administration using penetrants such as bile salts, fusidic acid, and other surfactants. In addition, oral administration is possible provided the polypeptide or other compound of the present invention can be formulated as an enteric coating or capsule. These compounds may be administered topically and / or localized in the form of an ointment, paste, gel or the like.

The required dosage range depends on the choice of peptide or other compound of the invention, the route of administration,
It depends on the nature of the formulation, the condition of the patient and the judgment of the physician. However, suitable dosages range from 0.1 to 100 μg / kg of patient weight. Given the variety of compounds available and the differing efficiencies of the routes of administration, the required dosage will vary widely. For example, oral administration would be expected to require higher dosages than intravenous administration. Such variation in dosage level can be adjusted using standard empirical optimization procedures, as is well understood in the art.

The polypeptide used for therapy can also be produced in the body of a patient in a therapy called “gene therapy” as described above. For example, cells from a patient
It is genetically engineered ex vivo by a polynucleotide such as DNA or RNA encoding the polypeptide, for example, using a retroviral plasmid vector. Thereafter, these cells are introduced into the patient.

[0067] Polynucleotide and polypeptide sequences provide a valuable source of information in identifying alternative sequences having similar homology. This is facilitated by storing such sequences in a computer readable medium and then using the stored data to search a sequence database with known search tools such as GCC.
Accordingly, in a further aspect, the present invention provides a computer readable medium having stored thereon a polynucleotide comprising the sequence of SEQ ID NO: 1 and / or a polypeptide encoded thereby.

The following definitions are provided to facilitate understanding of terms often used in the above description. As used herein, “antibody” includes polyclonal and monoclonal antibodies, chimeric antibodies, single-chain antibodies, humanized antibodies, as well as Fab fragments, including Fab or other products of an immunoglobulin expression library.

“Isolated” means altered “by the hand of man” from the natural state. If an "isolated" composition or substance occurs in nature, it has been changed or separated from its original environment, or both.
For example, a polynucleotide or polypeptide naturally occurring in the body of a living animal is not "isolated", but a polynucleotide or polypeptide separated from its naturally occurring co-localized material is described herein. As used herein, "isolated".

“Polynucleotide” generally refers to any polyribonucleotide or polydeoxyribonucleotide, which is unmodified RNA or DNA
Or modified RNA or DNA. “Polynucleotide” includes, but is not limited to, single- and double-stranded DNA, DNA in which single- and double-stranded regions are mixed, single- and double-stranded RNA, single-stranded RNA with mixed region and double-stranded region, hybrid molecule containing DNA and RNA (may be single-stranded or more typically double-stranded, where single-stranded and double-stranded regions are mixed May be included). In addition, "polynucleotide" may be RNA or D
NA or a triple-stranded region consisting of both RNA and DNA. The term "polynucleotide" also refers to DNA or RNA containing one or more modified bases.
And DNA having a backbone modified for stability or for other reasons
Also includes NA. "Modified" bases include, for example, tritylated bases and special bases such as inosine. Various modifications can be made to DNA and RNA. Thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides which are commonly found in nature, as well as DNA and RNA chemical forms characteristic of viruses and cells. . "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.

“Polypeptide” refers to a peptide or protein comprising two or more amino acids linked by a peptide bond or a modified peptide bond (ie, a peptide isostere). A "polypeptide" is a short chain (usually called a peptide, oligopeptide or oligomer) and a long chain (usually called a protein)
Refers to both. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. "Polypeptides" include amino acid sequences that have been modified by natural processes, such as post-translational processing, or by any of the chemical modification methods known in the art. Such modifications are elaborated in basic textbooks, more detailed scholarly articles and research literature. Modifications can be made anywhere in the polypeptide, including the peptide backbone, amino acid side chains, amino or carboxyl termini. The same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides can be branched for ubiquitination or cyclic, with or without branching. Cyclic, branched or branched cyclic polypeptides can result from post-translation natural processes and can also be produced by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent bonding of flavin, covalent bonding of heme moieties, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, covalent bonding of phosphatidylinositol. Bonds, crosslinks, cyclizations, disulfide bond formation, demethylation, covalent crosslink formation, cystine formation, pyroglutamate formation, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodine Transfer RNA-mediated addition of amino acids to proteins such as acetylation, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation, and ubiquitination (
For example, Proteins-Structure and Molecular Properties 2nd Ed., TE Cre
ighton, WH Freeman and Company, New York, 1993; Posttranslational Cova
lent Modification of Proteins, edited by BC Johnson, Academic Press, New York,
Wold, F., Post-translational Protein Modifications: Perspective in 1983
s and Prospects, pgs. 1-12; Seifter et al., “Analysis for protein modificati
ons and nonprotein cofactors ", Meth Enzymol (1990) 182: 626-646; and R
attan et al., “Protein Synthesis: Post-translational Modifications and Aging
", Ann NY Acad Sci (1992) 663: 48-62).

As used herein, a “variant” is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from a reference polynucleotide. Changes in the nucleotide sequence of this variant
The amino acid sequence of the polypeptide encoded by the reference polynucleotide may or may not be changed. Nucleotide changes can result in amino acid substitutions, deletions, additions, fusions and truncations of the polypeptide encoded by the reference sequence, as described below. A typical variant of a polypeptide differs in amino acid sequence from a reference polypeptide. In general, the sequences of the reference polypeptide and the variant are generally closely similar and limited to differences so that they will be identical in many regions. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, deletions, or additions in any combination. The amino acid residues to be substituted or added may or may not be those encoded by the genetic code. The variant of the polynucleotide or polypeptide may be a naturally occurring variant, such as an allelic variant, or a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides can be made by mutagenesis methods or by direct synthesis.

“Identity,” as known in the art, is a relationship between two or more polypeptide or polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between strands of such sequences. "Identity" can be calculated without difficulty by a known method,
Such methods include, for example, Computational Molecular Biology, Lesk, AM
, Oxford University Press, New York, 1988; Biocomputing: Informatics and
Genome Projects, Smith, DW, Academic Press, New York, 1993; Comput
er Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG
, Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology
, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribsk
ov, M. and Devereux, edited by J., M Stockton Press, New York, 1991; and Car
illo, H. and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), but not limited thereto. Methods for determining identity are designed to give the largest match between the sequences considered. In addition, methods for determining identity have been compiled into publicly available computer programs. Computer program methods for determining the identity between two sequences include the GCG program package (Devereux, J. et al., Nucleic Acids Research 12 (1): 387 (1984)), BL
ASTP, BLASTN and FASTA (Atschul, SF et al., J. Molec. Biol.
215: 403-410 (1990)). The BLAST X program is generally available from NCBI and other sources (BLAST Manual, Altsch
ul, S. et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S. et al., J. Mol. Biol.
215: 403-410 (1990)). The well-known Smith Waterman algorithm can also be used to determine identity.

The parameters for comparing polypeptide sequences include: 1) Algorithm: Needleman & Wunsch, J. Mol. Biol. 48: 443-453 (1970) Comparison matrix: BLOSSUM62, Hentikoff and Hentikoff, Proc. Natl. Acad.
Sci. USA, 89: 10915-10919 (1992) Gap penalty: 12 Gap length penalty: 4 A useful program with these parameters is Genetics Computer Group
(Madison WI) is generally available as a "gap" program. The above parameters are the default parameters for peptide comparison (default paramet
er) (no end gap penalty).

The parameters for comparing polynucleotide sequences include the following: 1) Algorithm: Needleman & Wunsch, J. Mol. Biol. 48: 443-453 (1970); Comparison matrix: match = + 10, mismatch = 0 Gap penalty: 50 Gap length penalty: 3 A useful program with these parameters is Genetics Computer Group
(Madison WI) as a "gap" program. These are the default parameters for nucleic acid comparison.

A preferred meaning for “identity” of polynucleotides and polypeptides is
Optionally provided in (1) and (2) below. (1) Specific examples of the polynucleotide comprise a polynucleotide sequence having at least 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the reference sequence of SEQ ID NO: 1. It further includes an isolated polynucleotide. Even if the polynucleotide sequence is the same as the reference sequence of SEQ ID NO: 1, with respect to the reference sequence,
It may contain up to an integer number of nucleotide variations. The mutation is selected from the group consisting of deletions, substitutions (including transitions and transversions) or insertions of at least one nucleotide, such mutations being at the 5 'or 3' terminal position of the reference nucleotide sequence, or at these terminal positions. Between the nucleotides in the reference sequence, individually or as one or more contiguous groups within the reference sequence. The number of nucleotide variations is determined by multiplying the total number of nucleotides of SEQ ID NO: 1 by the respective percent identity (divided by 100) and subtracting the product from the total number of nucleotides of SEQ ID NO: 1,
Following formula: _{n n} <x _n - can be obtained by (x _n · y). Wherein, n _n is the number of nucleotide alterations, x _n is the total number of nucleotides in SEQ ID NO: 1, y is about 0.70,80% for 0.60,70% for 0.60 for 60% for 50% 0.85, 90% for 0.80, 85%
Is 0.90 for 95%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%. • is a symbol representing a multiplicative operator, and the product of _xn and y is a non-integer. Round down to the nearest integer before subtracting from _xn . The alteration of the polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2 results in a nonsense, missense or frameshift mutation in the coding sequence, thereby altering the polypeptide encoded by the polynucleotide after such mutation. be able to.

By way of example, the polynucleotide sequence of the present invention is identical to the reference sequence of SEQ ID NO: 2.
Is 100% identical to the reference sequence or has a% identity of 1
Include up to an integer number of amino acid mutations relative to the reference sequence so that it is less than 00%.
Can be. The mutation may be a deletion, substitution (transition or deletion) of at least one nucleic acid.
And transversions) or insertions.
The mutation may be at the 5 'or 3' end of the reference polynucleotide sequence, or at
It may be anywhere between the terminal positions, individually or between the nucleic acids in the reference sequence.
It can intervene as one or more contiguous groups within a sub-array. Nucleic acid mutation
The number is determined by multiplying the total number of amino acids of SEQ ID NO: 2 by the percent identity value of each (divided by 100) and subtracting the product from the total number of amino acids of SEQ ID NO: 2.
And the following equation: n_n < x_n -(X_nY) can be obtained by Where n_nIs the number of amino acid mutations, x_nIs the array number
The total number of amino acids in No. 2 where y is 0.70 for 70%, for 80%
Is 0.80, for 85% is 0.85, etc. is a symbol representing the multiplication operator, x _n And the non-integer product of y is x_nRound down to the nearest integer before subtracting from
You.

(2) A specific example of the polypeptide is at least 50% of the reference sequence of SEQ ID NO: 2.
, 60, 70, 80, 85, 90, 95, 97 or 100% identity. The polypeptide sequence may be the same as the reference sequence of SEQ ID NO: 2, or may include up to an integer number of amino acid mutations with respect to the reference sequence. Such mutations are selected from the group consisting of deletions, substitutions (including conservative and non-conservative amino acid substitutions) or insertions of at least one amino acid, wherein the mutations are at the amino or carboxy terminal position of the reference polypeptide sequence. Or anywhere between these terminal positions, and may be interspersed individually between amino acids in the reference sequence, or as one or more contiguous groups within the reference sequence. The number of amino acid mutations is determined by multiplying the total number of amino acids in SEQ ID NO: 2 by the respective percent identity (divided by 100) and subtracting the product from the total number of amino acids in SEQ ID NO: 2, It is obtained by the following equation. _{n a <x a - (x} a · y) wherein, n _a is the number of amino acid alterations, x _a is the total number of amino acids in SEQ ID NO: 2, y is about 0.60 for 60% for 50% 0.60, 70% 0.70, 80%
0.80 for 85%, 0.85 for 85%, 0.90 for 90%, 0 for 95%.
0.97 for 95, 97% or 1.00 for 100%, is a symbol representing the multiplicative operator, and the product of non-integers of x _a and y is the closest approximation before subtracting the product from x _a Round down to an integer.

By way of example, the polypeptide sequence of the invention is identical to the reference sequence of SEQ ID NO: 2, ie has 100% identity to the reference sequence or has a% identity of 100%
It may include up to a certain integer number of amino acid mutations with respect to the reference sequence so as to be less than. Such mutations are selected from the group consisting of deletions, substitutions (including conservative and non-conservative amino acid substitutions) or insertions of at least one amino acid, wherein the mutations are at the amino or carboxy terminal position of the reference polypeptide sequence. Or anywhere between these terminal positions, and may be interspersed individually between amino acids in the reference sequence, or as one or more contiguous groups within the reference sequence.
The number of amino acid variations is determined by multiplying the total number of amino acids in SEQ ID NO: 2 by the respective percent identity (divided by 100) and subtracting the product from the total number of amino acids in SEQ ID NO: 2, ie, Required by n _a <x _a - in (x _a · y) formula, n _a is the number of amino acid alterations, x _a is the total number of amino acids in SEQ ID NO: 2, y 0.70,80% is about 70% for example 0.80 for 85% for 0.8%
And is a symbol representing a multiplicative operator, and the non-integer product of x _a and y is rounded down to the closest integer before subtracting the product from x _a .

“Fusion protein” refers to a protein encoded by two, often unrelated, fused genes or fragments thereof. As an example, EP-A-0 464
Describe fusion proteins comprising various portions of the constant region of an immunoglobulin molecule and other human proteins or portions thereof. In many cases, for use in therapy and diagnosis, it will be advantageous to use the immunoglobulin Fc region as part of a fusion protein, for example, to improve pharmacokinetic properties (eg, EP-A-0232). 262). On the other hand, for some uses
It may be desirable to remove the Fc portion after expressing, detecting and purifying the fusion protein.

All publications, including patents and patent application specifications, cited herein are incorporated by reference in their entirety, as if each publication was clearly and individually indicated. It shall be incorporated here.

In Northern blots of UT7-EPO cells, EPRG3Spt is observed as a 3 kb EPO-induced message. EPRG3Spt is induced by EPO in the presence of the protein synthesis inhibitor cycloheximide, but EPR is increased when cycloheximide alone is added.
No induction of G3Spt is seen. These observations indicate that EPRG3Spt is a primary EPO response gene, and that its induction is not dependent on new protein synthesis.
Therefore, induction of EPRG3Spt expression must be caused by direct activation of the signaling pathway by EPO. Stimulation with EPO alone did not produce detectable levels of the EPRG3Spt message, so EPO + cycloheximide treatment clearly showed EPRG3Spt treatment.
It leads to message-level superinduction. UT by hemin treatment
When erythroid differentiation of 7-EPO cells is induced, the expression of a 3 kb message is relatively decreased, and the expression of a smaller message is increased.

Examination of the cDNA amplified from human bone marrow cells by PCR revealed that when the cells were cultured with EPO + stem cell factor (SCF), expression of EPRG3Spt as a 1.1 kb size was induced, and after culturing with IL3 + SCF + flt3 ligand E to a lesser extent
It was shown that PRG3Spt was expressed. EPRG3Spt is not detected in untreated cells,
No detection was possible even after incubation with PO + SCF and G-CSF + SCF. Northern blots containing RNA from human bone marrow cells treated with the factor also showed strong induction of the 1.2 kb message of EPRG3Spt when EPO was included in the treatment of the cells.

By probing Northern blots containing RNA from other tissues, restricted expression of EPRG3Spt was seen. In addition, several different message sizes were found. The highest levels occurred in spleen (4.8, 4.2 kb), placenta (4.8, 0.9 kb) and peripheral blood leukocytes (0.9 kb).

Sequence Listing Free Text Sequence Information Sequence No. 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4

[0086]

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 C12P 21/02 C 15/09 ZNA C12Q 1/02 C12P 21/02 C12N 5/00 A C12Q 1/02 15/00 ZNAA (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), CA, JP (72) Dillon, Susan, Be. U.S.A. 19425 Pennsylvania, Chester Springs, Churamoa Circle 1209 F-term (reference) 4B024 AA01 AA11 BA80 CA04 CA09 DA02 DA05 DA11 EA04 GA11 GA18 GA19 HA14 HA15 HA17 4B063 QA01 QA07 QA17 QA18 QA19 QQ03 QRQ08 QRQQR QRQ QRQ QRQ QRQ QRQ QRQ QRQ QRQ QRQ QR QR QS36 QS38 QX01 QX07 4B064 AG27 CA10 CA20 CC24 DA01 DA13 4B065 AA19X AA26X AA49X AA53X AA60X AA72X AA90X AA93Y AB01 BA02 CA24 CA44 CA46 4H045 AA10 AA11 AA20 AA30 BA21 CA40 DA76 EA74

Claims (14)

[Claims] 1. An isolated polypeptide selected from the group consisting of: (i) at least (a) relative to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2 An isolated polypeptide comprising an amino acid sequence having 70% identity, (b) 80% identity, (c) 90% identity, or (d) 95% identity; ii) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or (iii) an isolated polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. 2. An isolated polynucleotide selected from the group consisting of the following (i) to (vi), or a nucleotide sequence complementary to the isolated polynucleotide: (i) SEQ ID NO: 2 Has at least (a) 70% identity, (b) 80% identity, (c) 90% identity, or (d) 95% identity to the amino acid sequence of SEQ ID NO: 2 over its entire length. An isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide; (ii) at least (a) 70% identity over its entire length to the nucleotide sequence encoding the polypeptide of SEQ ID NO: 2; (b) an isolated polynucleotide comprising a nucleotide sequence having 80% identity, (c) 90% identity, or (d) 95% identity; Sequence number over the entire length A nucleotide sequence having at least (a) 70% identity, (b) 80% identity, (c) 90% identity, or (d) 95% identity to the nucleotide sequence of (Iv) an isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO: 2, (vi) an isolated polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 1 A polynucleotide, and (vi) an isolated polynucleotide obtained by screening an appropriate library with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof under stringent hybridization conditions. 3. An antibody immunospecific for the polypeptide of claim 1. 4. A method of treating a subject, comprising: (i) when the subject requires increased activity or expression of the polypeptide of claim 1, (a) treating the polypeptide. Administering to said subject an effective amount of an agonist, and / or (b) producing an isolated polynucleotide comprising a nucleotide sequence encoding said polypeptide, wherein said polypeptide activity is produced in vivo. And (ii) when the subject requires suppression of the activity or expression of the polypeptide according to claim 1, (a) the polypeptide And / or (b) administering to the subject a nucleic acid molecule that suppresses the expression of the nucleotide sequence encoding the polypeptide. And / or administering to the subject a therapeutically effective amount of the polypeptide that competes with the polypeptide for its ligand, substrate, or receptor to the subject. Method. 5. A method for diagnosing a disease or susceptibility of a subject related to the expression or activity of the polypeptide according to claim 1 in the subject, the method comprising: (a) analyzing the genome of the subject; Examining for the presence of a mutation in the nucleotide sequence encoding the polypeptide of and / or (b) analyzing the presence or amount of expression of the polypeptide in a sample obtained from the subject A method comprising: 6. A screening method for identifying a compound that stimulates or suppresses the function of the polypeptide according to claim 1, comprising: (a) a candidate compound and the polypeptide (or a polypeptide carrying the polypeptide); Measuring the binding between the candidate compound and the fusion protein thereof using a label directly or indirectly bound to the candidate compound. (B) A candidate compound and the polypeptide (or the polypeptide). Measuring the binding to a peptide-bearing cell or its membrane) or its fusion protein in the presence of a labeled competitor; (c) a signal generated by the candidate compound upon activation or suppression of the polypeptide. Is examined using a detection system suitable for cells or cell membranes carrying the polypeptide, (d) a candidate compound and the polypeptide Preparing a mixture, measuring the activity of the polypeptide in the mixture and comparing the activity of the mixture to a standard; and (e) determining whether the candidate compound is Detecting the effect of the mRNA encoding the peptide and the polypeptide on the production of the polypeptide using, for example, an ELISA assay. 7. An agonist or antagonist of the polypeptide according to claim 1. 8. An expression system containing a polynucleotide capable of producing the polypeptide of claim 1 when the following expression system is present in a compatible host cell. 9. The host cell, under suitable culture conditions, produces a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2. A method for producing a recombinant host cell, comprising transforming or transfecting a cell with the expression system according to claim 8. 10. A recombinant host cell obtained by the method according to claim 9. 11. The recombinant host of claim 10, which expresses a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2. Cell membrane. 12. The method according to claim 10, comprising culturing the host cell according to claim 10 under conditions sufficient to produce said polypeptide, and recovering said polypeptide from said culture medium. Production method. 13. An isolated polynucleotide selected from the group consisting of the following (a) to (c):
An isolated polynucleotide comprising a nucleotide sequence having 70%, 80%, 90%, 95%, 97% identity, and (b) an isolated polynucleotide comprising the polynucleotide of SEQ ID NO: 3. A polynucleotide, or (c) the polynucleotide of SEQ ID NO: 3. 14. An isolated polynucleotide selected from the group consisting of: (a) at least 70% relative to the nucleotide sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4, An isolated polynucleotide comprising a nucleotide sequence having 80%, 90%, 95%, 97-99% identity; (b) an isolated polynucleotide comprising a polynucleotide of SEQ ID NO: 4 Or (c) the polynucleotide of SEQ ID NO: 4.