JP2003510076A - Human paralog of a head injury-induced cytoplasmic calcium-binding protein - Google Patents

Abstract

Abstract: ANIC-BP-2 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant methods are disclosed. Also disclosed are methods that utilize ANIC-BP-2 polypeptides and polynucleotides in diagnostic assays.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention encodes a human acute nerve-inducible calcium binding protein (ANIC-BP), hereinafter sometimes referred to as "ANIC-BP-2",
Relates to the polynucleotide concerned. The invention also provides expression vectors, host cells and antibodies. In addition, the invention also provides methods of producing the protein, and methods of treating or preventing disorders associated with expression of the protein. The invention further relates to inhibiting or activating the action of such polynucleotides and polypeptides.

BACKGROUND OF THE INVENTION Stroke, acute head trauma, multiple sclerosis and spinal cord injury are diseases for which there is no cure available so far. Stroke is the third leading cause of death and the burden on patients and social systems is enormous. In the case of ischemic stroke, which accounts for the majority of strokes, blockage of blood vessels in the brain is the first event. Head traumatic seizures are a predominant disease of the young in Western countries. The number of patients with hemorrhagic stroke due to mechanical shock and rupture of arteries is much smaller. A common feature is that licensed medicines are rarely available. Currently available therapeutic tools are based on pathophysiological ideas drawn from experimental studies of focal cerebral ischemia. These include pharmacological approaches aimed at arterial recanalization, inhibition of inflammatory processes, and protection of nerves. Further studies using the technique of arterial reperfusion are in the process. Initial clinical trials with polymorphonuclear leukocyte-dependent endothelial adhesion receptor antagonists are being completed, but the approach remains
Must be created. However, both approaches, which target only a single stage in the ischemic cascade, produce only limited benefits. Therefore, future treatments may best be based on combination therapy. The combination of low-dose acetylsalicylic acid (ASA) and sustained release dipyridamole has been shown to be an adjunct in preventing recurrence of stroke (Tijssen).
et al. , Int. J. Clin. Pract. , Suppl
． 91, 14-16, 1997). Another combination currently being planned for clinical evaluation is tPA plus an effective neuroprotective agent. But,
The results of these tests are far from being very effective. Thus, available for developing new medicines and establishing more broadly useful treatments for the treatment of patients suffering from acute nerve injury symptoms and diseases such as multiple sclerosis, There is an urgent need for further knowledge about pathophysiological mechanisms to provide medicinal targets.

The present invention focuses on Ca ^{2+ -binding} proteins, as there is considerable evidence for the role of Ca ^{2+ -binding} proteins in neuroprotection. Although the exact function of the calcium-binding protein (CaBP) is not completely known, CaB
P is proposed to have a buffering effect on intracellular Ca ²⁺ concentration. Ca ²⁺ overload activates a biochemical process that causes proteolysis and mitochondrial dysfunction, so this buffering capacity of CaBP may have a protective effect against stimulated toxic nerve damage (Heizmann et al., TINS
, 15, 259-64, 1992). There is extensive evidence to support this proposed role of CaBP in the regulation of Ca ²⁺ concentration and neuroprotection.

The major Ca ²⁺ -binding proteins (CaBP) (parvalbumin, calbindin-D28K, and calretinin) expressed in the central nervous system are highly specific and selective in various neuronal groups. It has various expression patterns. CaB
Of the P-expressing neurons, a few neurons express more than one major calcium binding protein, but most express only one type. Evidence is accumulating that the presence or absence of CaBP in certain cell types underlies a phenomenon known as selective vulnerability. Selective vulnerability is the property that certain types of neurons die in response to certain types of central nervous system (CNS) damage. For example, CA1 hippocampal neurons are selectively vulnerable to global ischemia, and cerebellar Purkinje cells are selectively vulnerable to head trauma, stroke, and fetal alcohol exposure. , Neurons in the substantia nigra are vulnerable to Parkinson's disease. Efforts have been made to correlate selective neuronal vulnerability with various CaBP expression patterns and one investigator found that high levels of CaBP were selectively vulnerable to injury. Reported to be found in one neuron group, while others have reported high levels of CaBP in neuron groups that are selectively resistant to injury. For example, neurons expressing high levels of parvalbumin have been reported to be selectively vulnerable to AMPA-induced toxicity (Weiss et al., Neurol., 40, 12).
88-1292, 1990) reported that cultured hippocampal neurons expressing high levels of calbindin-D28K are selectively resistant to glutamate-induced toxicity (Baimbridge et al. , TI
NS, 15, 303-8, 1992). Similarly, hippocampal neurons expressing high levels of calretinin are resistant to toxic doses of the excitotoxins glutamate, NMDA, kainate, and quisqualate (Winsky).
et al. , Novel Calcium-Binding Protei
ns, 277-300, 1991).

CaBP has also been shown to exhibit altered expression in a variety of CNS disease states, but also CaBP expression is associated with or selectively vulnerable to injury. The results are inconsistent regarding whether it is related to physical tolerance. Neurons expressing calbindin-D28K are associated with Alzheimer's disease (Iacopino et al., PNAS, 87, 4078-82, 1990, Hof et al., Exp. Neurol., 111).
, 293-301, 1991) and Huntington's disease (Kiyama et al.
al. , Brain Res. , 526, 303-07, 1990).
, It was reported to be selectively vulnerable to calbindin-D28 in the substantia nigra.
K-expressing neurons are not selectively vulnerable during Parkinson's disease (Ya
mada et al. , Brain Res. , 526, 303-07
, 1990). In the gerbil model of global ischemia, the presence of parvalbumin has been shown to be positively associated with survival in certain hippocampal cell types (Tortosa et al., Neurosci., 1, 3).
3-43, 1993), but in another study, hippocampal interneurons expressing parvalbumin were suggested to be selectively vulnerable during Alzheimer's disease (Brady et al., Neurosci., 80). , 1113-2
5, 1997).

[0006] Calbindin gene knockout mice suggest severe dysfunction in neurons that normally express this CaBP (eg, cerebellar Purkinje cells) despite the fact that these neurons appear morphologically normal. To indicate a functional loss (eg, ataxia). This finding suggests that CaBP is very important in the pattern of cellular activity (Airaksinen et al.,
PNAS, 94, 1488-93, 1997). In addition, calbindin-
Retroviral infection of motor neurons with D28k has been shown to have neuroprotective effects against IgG-induced toxicity from patients with amyotrophic lateral sclerosis (Ho et al., PNAS). , 93, 6796-8
01, 1996), and transfection of calbindin-D28k resulted in serum withdrawal, glutamate exposure, and toxicity due to the neurotoxin MPP +.
It has been shown to protect C12 cells (McMahon et al.,
Molec. Brain Res. , 526, 303-07, 1998.
).

In conclusion, there is considerable information regarding the role of calcium binding proteins in neurodegeneration. Although one CaBP exhibits a protective effect and another is certain to be responsible for selective vulnerabilities, the expression of specific CaBPs among different neuronal groups is different in such CaBPs. Whether it provokes a functional response remains unclear. Another finding is that the severity of different types of CNS injury can affect the apparent neuroprotective effect of CaBP. That is, some CaBPs confer resistance in injury models involving mild injury, but may not be able to buffer the Ca ²⁺ increase in more severe CNS injury. Thus, there is evidence to consider that CaBP confers resistance as well as vulnerability in the CNS injury process, but the mechanisms involved and regulation of response must be elucidated hereafter.

A gene family containing a functionally unidentified gene (MO25) has recently been isolated from a mouse-derived cDNA library (Miyamoto et al.,
Mol. Reprod. Dev. , 34, 1-7, 1993). This library was constructed from RNA isolated from early mouse embryos. Mo25
From the predicted amino acid sequence of Escherichia coli, the MO25 gene was found to have structural homology with the Ca ²⁺ -binding protein and lack the transmembrane domain. Suggests a possible involvement in development. However, the true function of this protein remains unclear. Another Mo25-like gene has been cloned from a Drosophila cDNA library (Nozaki).
et al. , DNA Cell Biol. , 15, 505-09,
1996). The deduced amino acid sequence of such Mo25 cDNA is mouse Mo2
It had 69.3% homology with the 5 homologue. The homologue in Saccharomyces cerevisiae was encoded in an open reading frame near the calcineurin B subunit gene. Recently, another gene was isolated from an Aspergillus hym A mutant strain (Karos et al., Mo.
l. Gen Genet. , 260, 510-521, 1999), yeast, plants, flies, helminths, fish, mice and humans. The intracellular function of the Hym protein has not been elucidated in any of the aforementioned organisms. Like many other proteins whose functional contributions are only partly understood, the drug discovery process currently includes "functional genomics," that is, high-throughput genomic or gene-based biology.
It is undergoing fundamental changes. This approach as a means of identifying genes and gene products that are therapeutic targets is rapidly replacing early approaches based on "positional cloning." A phenotype, which is a biological function or genetic disorder, is identified, which will then seek out the causative gene based on its location on the genetic map. Functional genomics relies heavily on high-throughput DNA sequencing techniques, as well as various bioinformatics tools to identify potentially interesting gene sequences from the many molecular biology databases currently available. There is. There is a continuing need to identify and characterize additional genes and their corresponding polypeptides / proteins as targets in drug discovery.

SUMMARY OF THE INVENTION The present invention relates to ANIC-BP-2, particularly ANIC-BP-2 polypeptides and ANIC-BP-2 polynucleotides, recombinants thereof and methods for their production. Such polypeptides and polynucleotides include, but are not limited to, stroke and acute head trauma, multiple sclerosis and spinal cord injury, a particular disorder hereafter referred to as "disease of the invention". Interested in the treatment of. In a further aspect, the present invention provides a method of identifying agonists as well as antagonists (eg, inhibitors) using the materials provided by the invention, as well as the compounds identified to cause an imbalance of ANIC-BP-2. A method of treating an associated condition. In yet another aspect, the invention features a diagnostic assay for detecting a disease associated with inappropriate ANIC-BP-2 activity or concentration.

[0010]     (Explanation of the invention)   In a first aspect, the invention relates to ANIC-BP-2 polypeptides.

Such a polypeptide comprises: (a) an isolated polypeptide encoded by a polynucleotide comprising the sequence of SEQ ID NO: 1; (b) at least 95% of the polypeptide sequence of SEQ ID NO: 2. , 96%
An isolated polypeptide comprising a polypeptide sequence having 97%, 97%, 98%, or 99% identity; (c) an isolated polypeptide comprising the polypeptide sequence of SEQ ID NO: 2; (d) At least 95%, 96% relative to the polypeptide sequence of SEQ ID NO: 2
, An isolated polypeptide having 97%, 98%, or 99% identity; (e) a polypeptide sequence of SEQ ID NO: 2; and (f) a polypeptide sequence of SEQ ID NO: 2 compared to 0. .95, 0.96, 0
． An isolated polypeptide having or comprising a polypeptide sequence having an identity index of 97, 0.98, or 0.99; (g) fragments and mutations of such polypeptides of (a)-(f). Contains the body.

The polypeptides of the present invention are considered to be ^* members of the calcium-binding protein family of polypeptides. Therefore, they are important as they can serve as novel pharmaceutical targets.

The biological properties of the ANIC-BP-2 are hereinafter referred to as “ANIC-BP-2 biological activity” or “ANIC-BP-2 activity”. Preferably, the polypeptides of the present invention exhibit at least one ANIC-BP-2 biological activity.

The polypeptides of the present invention also include variants of the aforementioned polypeptides, including all allelic forms as well as splice variants. Such a polypeptide is mutated from the reference polypeptide by insertions, deletions, and substitutions, whether conservative or non-conservative, or any combination thereof. Particularly preferred variants are some, eg 50 to 30, 30 to 20, 20.
To 10, 10 to 5, 5 to 3, 3 to 2, 2 to 1, or one amino acid has been inserted, substituted, or deleted in any combination.

A preferred fragment of the polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO: 2,
An isolated polypeptide comprising an amino acid sequence having at least 30, 50, or 100 consecutive amino acids, or at least 30, 50, or 100 consecutive amino acids from the amino acid sequence of SEQ ID NO: 2. Containing an isolated polypeptide comprising an amino acid sequence that has been deleted or deleted from the end. Preferred fragments are biologically active fragments which mediate the biological activity of ANIC-BP-2 and which have similar or improved activity, or
Includes reduced undesirable activity. Also preferred are fragments that are antigenic or immunogenic in animals, especially humans.

Fragments of the polypeptides of the present invention can also be used to produce the corresponding full length polypeptides by peptide synthesis. Therefore, these variants can be used as intermediates for producing the full-length polypeptides of the invention. The polypeptide of the invention may be in the form of a "mature" protein, or
It can be part of a larger protein, such as a precursor or fusion protein. Includes additional amino acid sequences, including secretory or leader sequences, prosequences, sequences utilized for purification, eg, multi-histidine residues, or additional sequences for stabilization during recombinant production. And, it is often advantageous.

The polypeptides of the present invention were isolated, for example, from natural sources, from genetically engineered host cells containing an expression system (see below), or using, for example, an automated peptide synthesizer. , Chemical synthesis, or a combination of such methods, etc., and may be prepared by any compatible method. Means for preparing such polypeptides are well understood in the art.

In a further aspect the invention relates to a polynucleotide of ANIC-BP-2. Such a polynucleotide comprises: (a) at least 95%, 9% of the polynucleotide sequence of SEQ ID NO: 1;
An isolated polynucleotide comprising a polynucleotide sequence having 6%, 97%, 98%, or 99% identity; (b) an isolated polynucleotide comprising the polynucleotide of SEQ ID NO: 1; ) At least 95%, 96% of the polynucleotide of SEQ ID NO: 1
, Isolated polynucleotide having 97%, 98%, or 99% identity; (d) an isolated polynucleotide of SEQ ID NO: 1; (e) at least 95 to the polypeptide sequence of SEQ ID NO: 2. %, 96%
, An isolated polynucleotide comprising a polynucleotide sequence encoding a polypeptide sequence having 97%, 98%, or 99% identity; (f) a polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2; (G) at least 95%, 96% relative to the polypeptide sequence of SEQ ID NO: 2.
An isolated polynucleotide having a polynucleotide sequence that encodes a polypeptide sequence having 97%, 98%, or 99% identity; (h) an isolated polynucleotide that encodes the polypeptide of SEQ ID NO: 2; i) 0.95, 0.96 compared to the polynucleotide sequence of SEQ ID NO: 1
An isolated polynucleotide having or comprising a polynucleotide sequence having an identity index of 0.97, 0.98, or 0.99; (j) as compared to the polypeptide sequence of SEQ ID NO: 2. , 0.95, 0.96, 0
． An isolated polynucleotide having or comprising a polynucleotide sequence encoding a polypeptide sequence having an identity index of 97, 0.98, or 0.99; and a fragment or variant of said polynucleotide. Or a polynucleotide that is complementary to the aforementioned polynucleotide over its entire length.

A preferred fragment of the polynucleotide of the present invention is an isolated polynucleotide comprising a nucleotide sequence having at least 15, 30, 50 or 100 bases consecutive from the sequence of SEQ ID NO: 1, or An isolated polynucleotide comprising a sequence that is contiguous, at least 30, 50, or 100 bases removed or deleted from the sequence of SEQ ID NO: 1.

Preferred variants of the polynucleotides of the present invention include polymorphisms, including splice variants, allelic variants, as well as polynucleotides having one or more single nucleotide polymorphisms (SNPs). It also includes.

The polynucleotide of the present invention also comprises the amino acid sequence of SEQ ID NO: 2, and also includes some of them, eg 50 to 30, 30 to 20, 20 to 10, 10 to 5 Also included are polynucleotides encoding polypeptide variants in which 5 to 3, 3 to 2, 2 to 1, or 1 amino acid residues have been replaced, deleted, or added in any combination.

In a further aspect, the invention also provides a polynucleotide that is an RNA transcript of a DNA sequence of the invention. Accordingly, (a) comprises an RNA transcript of a DNA sequence encoding the polypeptide of SEQ ID NO: 2; (b) is an RNA transcript of a DNA sequence encoding the polypeptide of SEQ ID NO: 2; (c An RNA transcript of the DNA sequence of SEQ ID NO: 1, or (d) an RNA transcript of the DNA sequence of SEQ ID NO: 1; and an RNA polynucleotide complementary to them Nucleotides are provided.

The polynucleotide sequence of SEQ ID NO: 1 is Hym A (Nozaki et al.
al. , DNA cell Biol. , 15, 505-09, 19
96) and Mo25 (Karos et al., Mol. Gen Ge.
net. , 260, 510-521, 1999). The polynucleotide sequence of SEQ ID NO: 1 is a cDNA sequence encoding the polypeptide of SEQ ID NO: 2. The polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2 may be identical to the polypeptide coding sequence of SEQ ID NO: 1, or, as a result of duplication of the genetic code (degeneracy), also SEQ ID NO: 2. A sequence other than SEQ ID NO: 1 which encodes the polypeptide of The polypeptide of SEQ ID NO: 2 is related to other proteins of the ^* calcium binding protein family, which have homology and / or structural similarity with Hym A and Mo25 proteins.

Preferred polypeptides of the invention, as well as polynucleotides, are expected to have, inter alia, similar biological functions / properties to their homologous polypeptides and polynucleotides. Furthermore, preferred polypeptides of the invention, as well as polynucleotides, have at least one ANIC-BP-2 activity.

The polynucleotide of the present invention is a cDNA of mRNA derived from cells of the human central nervous system.
It can be obtained from the library using standard cloning and screening techniques (eg, Sambrook et al., Molcu.
Lar Cloning: A Laboratory Manual, 2n
d Ed. , Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.M. Y. (19
89)). The polynucleotides of the present invention can be obtained from natural sources such as genomic DNA libraries, or can be synthesized using well known and commercially available techniques.

When the polynucleotide of the present invention is used for recombinant production of the polypeptide of the present invention,
The polynucleotide may comprise the sequence encoding the mature polypeptide, or may include only that, or may encode a leader or secretory sequence, a pre, or pro or preproprotein sequence, or other fusion peptide moiety. etc,
Within the open reading frame may be the coding sequence for the mature polypeptide, along with other coding sequences. For example, a marker sequence can be encoded that facilitates purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker sequence is provided in the pQE vector (Qiagen, Inc.) and is described in Gentz et al. , Proc. Natl. Acad. Sci
． USA (1989) 86: 821-824, which is a hexa-histidine peptide or HA tag. The polynucleotide may also include non-coding 5'- and 3'sequences such as transcribed / untranslated sequences, splicing and polyadenylation signals, ribosome binding sites, and mRNA stabilizing sequences. Good.

A polynucleotide which is identical to the polynucleotide sequence of SEQ ID NO: 1 or which has sufficient identity is used as a hybridization probe for cDNA as well as genomic DNA, or as a nucleic acid amplification reaction (eg PCR). )
It can be used as a primer for. Such probes and primers were used to isolate full-length cDNA and genomic clones encoding the polypeptides of the invention, as well as having high sequence similarity to SEQ ID NO: 1, typically at least 95% homology. Of other genes, including paralogs from human origin, orthologs and paralogs from non-human species, having
It can be used to isolate clones. Preferred probes and primers generally comprise at least 15 bases, preferably at least 30 bases and may have at least 50 if not at least 100 bases. Particularly preferred probes can have 30-50 bases.
Particularly preferred primers can have 20 to 25 bases.

A polynucleotide encoding a polypeptide of the invention, including homologues from a species other than human, is a sequence of SEQ ID NO: 1 or a fragment thereof, preferably under at least 15 bases, under stringent hybridization conditions. May be obtained by a method comprising a step of screening a library with a labeled probe having: and a step of isolating a full-length cDNA and a genomic clone containing the polynucleotide sequence. Such hybridization techniques are well known to those of ordinary skill in the art. Suitable and stringent hybridization conditions are: 50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, And incubating in a solution containing 20 micrograms / ml denatured and cleaved salmon sperm DNA at 42 ° C. overnight; followed by washing the filters in 0.1 × SSC at about 65 ° C. .
Thus, the invention is obtained by screening a library under stringent hybridization conditions, preferably with at least 15 bases, a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof, preferably at least 100. It also includes an isolated polynucleotide having a sequence of bases.

Those skilled in the art will often be aware that the isolated cDNA sequence is often incomplete and the region encoding the polypeptide does not extend completely to the 5'end. . This is because the enzyme "reactivity" (a measure of the ability of the enzyme to remain bound to the template during the polymerization reaction) is inherently low; It is the result that the DNA replication of the template mRNA could not be completed.

Several methods are available and well known to those skilled in the art for obtaining full-length cDNAs or extending short cDNAs, for example, rapid amplification of cDNA ends (
Some are based on the RACE method (see, for example, Frohman et al.,
Proc. Nat. Acad. Sci. USA 85, 8998-9.
002, 1988). For example, the Marathon ™ method (Clon
tech Laboratories Inc. ), A recent improved method significantly simplified the search for longer cDNAs. In the Marathon ™ method, cDNA is prepared from mRNA extracted from selected tissues and a “adapter” sequence is ligated to each end. The DNA is then sequenced using a combination of gene-specific as well as adapter-specific oligonucleotide primers.
Nucleic acid amplification (PCR) is performed to amplify the "missing"5'end of. Then
An “nested” primer, ie, a primer designed to anneal within the amplification product (typically an adapter-specific primer that anneals further 3 ′ to the adapter sequence, and a known gene sequence). 5 '
The PCR reaction is repeated with a gene-specific primer that anneals to the side. The product of this reaction is then analyzed by DNA sequencing and the product ligated directly to the existing cDNA to obtain the complete sequence, or new sequence information for the design of the 5'primer. Utilizing this, a full-length cDNA can be constructed by performing a separate full-length PCR.

Recombinant polypeptides of the invention can also be prepared from genetically engineered host cells that contain an expression system by methods well known in the art. Thus, in a further aspect, the present invention provides expression systems comprising one or more polynucleotides, host cells genetically engineered to contain such expression systems, as well as recombinant production of the polypeptides of the invention. Regarding Cell-free translation systems can also be used to produce such proteins using RNA derived from the DNA constructs of the invention.

For recombinant production, host cells may also be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention. Polynucleotides are described by Davis et al. , Basic Methods in Mole
circular Biology (1986) and Sambrook et a.
l. It can be introduced into host cells by methods described in many standard laboratory manuals, such as (Id.). Preferred methods for introducing a polynucleotide into a host cell include, for example, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid mediated transfection, electroporation, transduction, scraping. • Includes loading, ballistic introduction or infection.

Representative examples of suitable hosts include bacterial cells such as streptococci, staphylococci, E. coli, actinomycetes, and Bacillus subtilis cells, fungal cells such as yeast cells and Aspergillus cells, Drosophila S2 and Spodoptera litura Sf9 cells. Insect cells, CHO, COS,
Animal cells such as HeLa, C127, 3T3, BHK, HEK293 and Bowes melanoma cells, as well as plant cells are included.

A variety of expression systems, such as chromosomal, episomal and viral derived systems,
For example, bacterial plasmid origin, bacteriophage origin, transposon origin,
Yeast episome, insertion factor, yeast chromosomal factor, baculovirus,
Papovaviruses such as SV40, vectors derived from viruses such as vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and retrovirus, and those derived from plasmids and bacteriophage genetic factors such as cosmids and phagemids. , Vectors derived from combinations thereof can be used. The expression system may include control regions that induce and at the same time regulate expression. Generally, a polynucleotide can be maintained, augmented, or expressed for the production of the polypeptide in a host,
Any system or vector can be used. Such suitable polynucleotide sequences are described, for example, in Sambrook et al., Supra. It may also be inserted into the expression system by any of a variety of well-known and conventional techniques, such as those shown in (Id.). Appropriate secretion signals may also be incorporated into the desired polypeptide to allow secretion of the translated protein into the endoplasmic reticulum lumen, the periplasmic space, or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals.

When the polypeptides of the invention are to be expressed for use in screening assays, it is generally preferred that the polypeptides be produced on the cell surface. The cells may then be harvested prior to use in the screening assay. When the polypeptide is secreted into the medium, the medium can be recovered to recover and purify the polypeptide. When produced intracellularly, the cells must be prelysed before the polypeptide can be recovered.

Polypeptides of the invention may be ammonium sulfate or ethanol precipitated, acid extracted,
Anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography,
Recombinant cell cultures can be harvested and purified by well-known methods, including hydroxylapatite chromatography, and lectin chromatography. Most preferably, high performance liquid chromatography is used for purification. When the polypeptide is denatured during intracellular synthesis, isolation and / or purification, well known techniques for protein refolding can also be used to regenerate the active conformation.

The polynucleotide of the present invention can be used as a diagnostic reagent by detecting a mutation in a related gene. Detecting a mutant form of a gene identified by the polynucleotide of SEQ ID NO: 1 in a cDNA or genomic sequence, which is associated with dysfunction, can be detected as underexpression, overexpression, or localization of the gene. Or a disease caused by time-shifted expression,
Alternatively, diagnostic tools can be provided that can supplement or confirm the diagnosis of susceptibility to disease. Individuals carrying mutations in the gene may be detected at the DNA level by various techniques well known in the art.

Nucleic acid for diagnosis can be obtained from cells of a subject such as blood, urine, saliva, tissue biopsy or autopsy material. The genomic DNA can be used for direct detection, or it can be enzymatically amplified using PCR, preferably RT-PCR, or other amplification techniques prior to analysis. RNA or c
DNA can also be used in a similar form. Deletions and insertions can be detected by changes in the size of their amplification products as compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled ANIC-BP-2 base sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion, or by differences in melting temperatures. DNA sequence differences may be due to changes in electrophoretic mobility of DNA fragments on gels with or without denaturing agents, or direct DNA sequencing (eg, Myers et al., Science (
1985) 230: 1242). Sequence changes at specific sites may be due to nuclease protection assays, such as RNase and S1 protection, or chemical cleavage methods (Cotton et al., Proc. Na.
tl. Acad. Sci. USA (1985) 85: 4397-44.
01)).

Arrays of oligonucleotide probes comprising the ANIC-BP-2 polynucleotide sequence or fragments thereof can also be constructed, eg, to perform an effective screen for genetic mutations. Such arrays are preferably high density arrays or grids. The methods of arraying technology are well known and have general application, and can be used to answer various questions in molecular genetics, including gene expression, gene linkage, and genetic variability, for example: , M .; Ch
ee et al. , Science, 274, 610-613 (1996).
) And other references cited therein.

Detection of an abnormal increase or decrease in the expression level of a polypeptide or mRNA is
The susceptibility of a subject to the diseases of the invention can also be used to diagnose or determine. Decreased or elevated expression can be achieved, for example, by nucleic acid amplification, eg PCR, R
It can be measured at the RNA level using any of the methods well known in the art for quantifying polynucleotides, such as T-PCR, RNase protection, Northern blotting, and other hybridization methods. . Assay techniques that can be used to quantify protein levels, such as a polypeptide of the invention, in a sample derived from a host are well known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis, and ELISA assays.

Accordingly, in another aspect, the invention provides: (a) a polynucleotide of the invention, preferably the nucleotide sequence of SEQ ID NO: 1, or a fragment or RNA transcript thereof; (b) of (a) A nucleotide sequence complementary to (c) the polypeptide of the present invention, preferably the polypeptide of SEQ ID NO: 2, or a fragment thereof, or (d) the polypeptide of the present invention, preferably the polypeptide of SEQ ID NO: 2. It relates to a diagnostic kit comprising an antibody.

It will be appreciated that in any such kit, (a), (b), (c), or (d) may constitute a substantial component.
Such kits will be useful in diagnosing a disease or susceptibility to a disease, especially among others, the diseases of the invention.

The polynucleotide sequences of the present invention are useful for chromosome location analysis studies. The sequences are capable of specifically targeting and hybridizing to specific locations on individual human chromosomes. Mapping of related sequences to chromosomes in accordance with the present invention is of primary importance in relating those sequences to gene-related disorders.
It is a stage. Once the sequence is mapped to the correct chromosomal location, the physical location of the sequence on the chromosome can be correlated with the gene map data. Such data is, for example, V.I. McKusick's Mendelian Inheritance in Man (John)
s Hopkins University Welch Medical L
(available online from ibrary). Then, the relationship between the gene mapped to the same chromosomal region and the disease is identified through a linkage group analysis (co-inheritance of physically adjacent genes). The exact position on the human chromosome for a genomic sequence (such as a gene fragment) is determined by radiation hybrid (RH) mapping (W
alter, M.A. , Spilllet, D .; , Thomas, P .; , Wei
ssenbach, J .; , And Goodfellow, P .; , (199
4) Method for constructing radiation hybrid map of whole genome (A method f
or constructing radiation hybrid map
s of whole genomes), Nature Genetics
7, 22-28). Some RH panels, such as the GeneBridge4 RH panel (Hum. Mol. G.
enet. 1996 Mar; 5 (3): 339-46 Radiation hybrid map of the human genome.
f the human genome); Gyapay G .; , Schm
itt K. , Fizames C .; , Jones H. , Vega-C
zarry N.V. , Spillett D .; , Muselet D .; , P
rud'Home J. F. , Dib C .; , Affray C. ，
Morrisette J. Weissenbach J. , Goodf
ellow P. N. ) Is available from Research Genetics (Huntsville, AL, USA). Using this panel, 93 PCRs are performed with primers designed from the gene of interest on RH DNA to determine the chromosomal location of the gene. Each of these DNAs contains a random human genomic fragment maintained in a hamster background (human / hamster hybrid cell line). These PCR
Gives a score of 93 indicating the presence or absence of the PCR product of the gene of interest. These scores are compared to the scores generated using PCR products from genomic sequences of known position. This comparison can be found at http: // www. genome. wi. m
it. Do it with edu. The gene of the present invention has the position 13 (D13S1
68-D13S153) ^* .

The polynucleotide sequences of the present invention are also useful tools for studying tissue expression. Such studies allow the determination of the expression pattern of the polynucleotides of the invention, which can give an indication as to the expression pattern of the encoded polypeptide in a tissue by detection of the mRNA encoding it. . The techniques used are well known in the art and include cDNA microarray hybridization (Schena et al., Science, 270,
467-470, 1995 and Shalon et al. , Genom
e Res. , 6, 639-645, 1996), and in situ hybridization methods for clones arranged on a grid, as well as nucleotide amplification methods such as PCR. A preferred method utilizes the TAQMAN ™ method available from Perkin Elmer. The results of these studies can also give an indication of the normal function of the polypeptide in that organism. In addition, m
A comparison of the normal expression pattern of RNA with the expression pattern of RNA encoded by a variant form of the same gene (eg, having a change in the polypeptide encoding a potential or regulatory mutation), Role of the polypeptide of the invention,
Alternatively, it can also provide useful insights into the role of its inappropriate expression during disease. Such inappropriate expression may be characteristic in time, space or simply quantitatively.

Polypeptides of the invention include adrenals, brain, heart, kidneys, liver and spleen, lymph nodes,
Soares melanocyte 2NbHM, Soares retina N2b4HR
, Testicular, umbilical vein, colon, parathyroid tumor, pineal gland, uterus, pancreas, aorta, eye, whole embryo, tonsil, parathyroid gland, foreskin, stomach (NB by electronic northern analysis)
).

A further aspect of the invention relates to antibodies. The polypeptides of the present invention or fragments thereof, or cells expressing them can be used as an immunogen for generating immunospecific antibodies against the polypeptides of the present invention. The term "immunospecific" means that the antibody has a substantially higher affinity for a polypeptide of the invention than its affinity for other related polypeptides in the prior art.

The antibody raised against the polypeptide of the present invention is administered to the animal, preferably a non-human animal, using the usual protocol, the fragment carrying the polypeptide or the epitope, or the cell. It can be obtained. For preparation of monoclonal antibodies, any technique which provides antibodies produced by subculturing cell lines can be used. Examples thereof include hybridoma method (Kohler, G. and Milstein, C., Nature (1975) 256: 495-497), trioma method, human B cell hybridoma method (Kozbor et al., Immunology Toda).
y (1983) 4:72) and the EBV hybridoma method (Cole et.
al. , Monoclonal Antibodies and Canc
er Therapy, 77-96, Alan R. et al. Liss, Inc.
, 1985).

Techniques for making single chain antibodies, such as those described in US Pat. No. 4,946,778, can be applied to make single chain antibodies to the polypeptides of the invention. Similarly, transgenic mice, or other organisms including other mammals, may be utilized for expression of humanized antibodies.

The above-mentioned antibody is used for isolation or identification of a clone expressing the polypeptide, or for purification of the polypeptide by affinity chromatography.
It can also be used. Antibodies against the polypeptides of the invention can also be used, inter alia, to treat the diseases of the invention.

The polypeptides and polynucleotides of the present invention can also be used as vaccines. Accordingly, in a further aspect, the present invention is a method for inducing an immune response in a mammal that protects the animal from the disease, whether or not the disease is already chronic in the individual. Antibody and / or
For example, it relates to a method comprising inoculating said mammal with a polypeptide of the invention suitable for generating a T cell immune response comprising cytokine producing T cells or cytotoxic T cells. An immune response in a mammal is for the purpose of inducing such an immune response to produce antibodies in order to protect said animal from the diseases of the present invention,
It can also be elicited in vivo by a method comprising delivering a polypeptide of the invention via a vector that encodes the polypeptide and directs expression of the polynucleotide. One means of administering the vector is by facilitating its introduction into the desired cells as a coating on particles or the like. Such nucleic acid vector may be DNA, RNA, modified nucleic acid, or
It may be composed of a DNA / RNA hybrid. For use in vaccines, the polypeptide or nucleic acid vector will usually be provided as a vaccine formulation (composition). The formulation may further comprise a suitable carrier.
Since the polypeptide may be decomposed in the stomach, it is preferably administered parenterally (eg, subcutaneous, intramuscular, intravenous, or intradermal injection). Formulations suitable for parenteral administration may include antioxidants, buffers, bacteriostats, and sterile aqueous and non-aqueous injections that may contain solutes that render the formulation isotonic with the blood of the recipient. Solutions; and sterile aqueous and non-aqueous suspensions, which may contain suspending agents or thickening agents. The formulations may also be presented in unit-dose or multi-dose containers, for example, sealed ampoules or vials, and lyophilized, merely by addition of a sterile liquid carrier immediately before use. It can also be saved in the state. The vaccine formulation may also include adjunct systems to enhance the immunogenicity of the formulation, such as oil-in-water systems as well as other systems known in the art. The dose depends on the specific activity of the vaccine and can be easily determined by routine experimentation.

The polypeptides of the present invention have one or more biological functions associated with one or more disease states, in particular the inventive diseases described herein above. Therefore, it is useful to identify compounds that stimulate or inhibit the function or concentration of the polypeptide. Thus, in a further aspect, the invention provides a method of screening compounds to identify those which stimulate or inhibit the function or concentration of said polypeptide. Such methods identify agonists or antagonists that can be used for the purpose of treating and preventing the diseases of the invention as described herein above. Compounds can be identified from a variety of sources, eg, cells, cell-free preparations, chemical libraries, collections of chemical compounds, and mixtures of natural products. The agonists or antagonists thus identified may optionally be natural or modified substrates, ligands, receptors, enzymes, etc. of the polypeptide; their structural or functional mimetics (Coligan et al., Current Protocols i
n Immunology 1 (2): Chapter 5 (1991)) or a small molecule.

In the screening method, the binding of a candidate compound to the polypeptide, a cell or membrane presenting the polypeptide, or a fusion protein thereof is determined by a label directly or indirectly bound to the candidate compound. , May be simply measured.
Alternatively, the screening method may also measure or (qualitatively or quantitatively) detect competitive binding of a candidate compound to a polypeptide against a labeled competitor (eg agonist or antagonist). included. Furthermore, these screening methods use a detection system suitable for cells displaying the polypeptide to examine whether or not the candidate compound causes a signal generated by activation or inhibition of the polypeptide. Good. Inhibitors of activation are generally
Assay in the presence of a known agonist and observe the effect of the presence of the candidate compound on activation by the agonist. Furthermore, in the screening method, in order to form a mixture, a step of mixing a candidate compound with a solution containing the polypeptide of the present invention, a step of measuring ANIC-BP-2 activity in the mixture, and an ANIC-of the mixture. Comparing the BP-2 activity with a control mixture containing no candidate compound.

The polypeptides of the present invention can also be used in conventional low throughput screening methods and also in high throughput screening (HTS) formats. Such HTS format is a 96-hole, and more recently,
The well-established use of 384-well microtiter plates as well as Sch
ullek et al. , Anal. Biochem. , 246, 2
0-29, (1997) and the emerging methods such as the nanowell method.

As previously described herein, fusion proteins, such as those made from Fc proteins and ANIC-BP polypeptides, may be used in high throughput screening assays to identify antagonists to the polypeptides of the invention. Can also be used (D. Bennett et al., J. M.
ol. Recognition, 8: 52-58 (1995); and
K. Johnson et al. , J. Biol. Chem. , 2
70 (16): 9459-9471 (1995)). Screening Procedures The polynucleotides, polypeptides and antibodies to the polypeptides of the present invention can also be used to form screening methods to detect the effect of added compounds on the production of mRNAs and polypeptides in cells. For example, monoclonal assays as well as polyclonal antibodies can be used to construct an ELISA assay to measure the concentration of secreted or cell associated polypeptides by standard methods known in the art. It can be used to discover substances (also called antagonists or agonists, respectively) that inhibit or enhance the production of the polypeptide from appropriately engineered cells or tissues.

The polypeptides of the present invention can also be used to identify either membrane bound or soluble receptors by standard receptor binding techniques known in the art. . These include, but are not limited to, labeling the polypeptide with a radioisotope (eg, ¹²⁵ I), chemically modifying (eg, biotinylation), or peptides suitable for detection or purification. Includes ligand binding as well as cross-linking assays that fuse with sequences and incubate with putative receptor sources (cells, cell membranes, cell supernatants, tissue extracts, body fluids). Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy. These screening methods, whether for agonists and antagonists of the polypeptide, that compete with the binding of the polypeptide to its receptor,
It can also be used to identify. Standard methods for performing such assays are well understood in the art.

Examples of the antagonist of the polypeptide of the present invention include an antibody or, in some cases, an oligonucleotide or protein closely related to a ligand, substrate, receptor, enzyme, etc. of the polypeptide, Some include fragments of ligands, substrates, receptors, enzymes, etc .; or small molecules that bind to a polypeptide of the invention but do not elicit a reaction, thus preventing the activity of the polypeptide.

Screening methods can also include the use of transgenic technology and the ANIC-BP-2 gene. Techniques for constructing transgenic animals are well established. For example, the ANIC-BP-2 gene is an embryo genetically modified by microinjection into a male nucleus of a fertilized oocyte, retrovirus introduction into an embryo before or after transplantation, or electroporation. It can be introduced by injecting stem cells into the host blastocyst. Particularly useful transgenic animals are so-called "knock-in" animals in which the animal's gene is replaced in its animal genome by its human equivalent gene. Knock-in transgenic animals are useful for target confirmation in the drug discovery process, where the compound is specific for human targets. Other useful transgenic animals are so-called "knocks", in which the expression of an ortholog of the polypeptide of the invention is partially or completely inactivated, which is encoded by an intracellular endogenous DNA sequence.・ It is an "out" animal. Gene knockout may be targeted to a particular cell or tissue, may be performed only in a particular cell or tissue as a result of technical limitations, or may be all in an animal. Alternatively, it may be performed in substantially all cells. The technology of transgenic animals is also
The introduced gene is expressed to provide large amounts of the polypeptide of the invention,
A whole animal expression-cloning system is provided.

Screening kits for use in the methods described above form a further aspect of the invention. Such a screening kit comprises (a) the polypeptide of the present invention; (b) a recombinant cell that expresses the polypeptide of the present invention; (c) a cell membrane that expresses the polypeptide of the present invention; or (d) the present invention. It is preferred that the polypeptide comprises any of the following: an antibody against the polypeptide; wherein the polypeptide is of SEQ ID NO: 2.

In any such kit, (a), (b), (c) or (d)
It will be appreciated that it constitutes a substantial component. (Terms) The following definitions are provided to promote understanding of specific terms that are already frequently used.

As used herein, “antibody” includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments that include the products of Fab or other immunoglobulin expression libraries.

“Isolated” means altered “by the hand of man” from its natural state, ie, if naturally occurring, altered from its original environment, or
Or taken out, or both. For example, a polynucleotide or polypeptide that naturally occurs in a living organism is not "isolated", but the same polynucleotide or polypeptide separated from its naturally occurring coexisting material is According to the use of the term, "isolated".
Furthermore, a polynucleotide or polypeptide introduced into an organism by transformation, genetic engineering or any other recombinant method is "isolated even if it is still present in said organism. The creature may or may not be alive.

“Polynucleotide” generally refers to either polyribonucleotides (RNA) or polydeoxyribonucleotides (DNA), which may be unmodified or modified RNA or DNA. A "polynucleotide" is, without limitation, a mixture of single and double stranded DNA, single and double stranded regions D
NA, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, may be single-stranded, or more typically double-stranded or single-stranded and double-stranded regions A hybrid molecule comprising DNA and RNA, which may be a mixture of In addition, "polynucleotide" refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term "polynucleotide" refers to DNA or RNA containing one or more modified bases and DNA or R having a backbone modified for stability or other reasons.
Including NA. "Modified" bases include, for example, tritylated bases as well as unusual bases such as inosine. Various modifications may be made to DNA and RNA, and thus, a "polynucleotide" is a chemically, enzymatically, or metabolically modified form of a polynucleotide typically found in nature. , And chemical forms of DNA and RNA that are characteristic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.

By “polypeptide” is meant any polypeptide comprising two or more amino acids linked to each other by a peptide bond or a modified peptide bond, ie, a peptide isostere. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, as well as long chains, commonly referred to as proteins. Polypeptides can contain amino acids other than the 20 amino acids of the genetic code. "Polypeptide" also includes amino acid sequences modified by natural processes such as post-translational processing, or by chemical modification methods well known in the art. Such modifications are well described in basic texts and more detailed monographs, as well as in many research literature. Modifications can be made anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Similarly, a given polypeptide may contain many types of modifications. Polypeptides may be branched, as a result of ubiquitination, and may be ring structures, with or without branching. Cyclic, branched, as well as branched cyclic polypeptides may result from post-translational natural processes or may be produced synthetically. Modifications include acetylation, acylation, ADP-ribosylation,
Amidation, biotinylation, covalent bond of flavin, covalent bond of heme moiety, covalent bond of nucleotide or nucleotide derivative, covalent bond of lipid or lipid derivative, covalent bond of phosphotidylinositol, cross linking, cyclization, disulfide bond. Formation, demethylation, covalent cross-link formation, cystine formation, pyroglutamate formation, formylation, γ-carboxylation, glycosylation, GP
I. Anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic treatment, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation, etc. Transfer RNA-mediated amino acid addition to proteins , And ubiquitination (eg, protein-structure and molecular properties, Second Edition (P
proteins-Structure and Molecular Prop
erties, 2nd Ed. ), T.S. E. Creighton, W.M. H
． Freeman and Company, New York, 1993.
Post-translational covalent modification of proteins (Post-translational Cov
alent Modification of Proteins), B.I. C
． Johnson Ed. , Academic Press, New Y
Ork's Wold, F.M. , Post-translational protein modification: Future perspectives and expectations (Post
-Translational Protein Modifications
: Perspectives and Prospects), 1-12; S
eifter et al. "Analysis of protein modification and non-protein cofactors (A
analysis for protein modifications an
d nonprotein cofactors, "Meth. Enzy
mol. , 182, 626-646, 1990, and Rattan.
et al. , "Protein synthesis: post-translational modification and aging (Protein
Synthesis: Post-translational Modular
ations and Aging) ", Ann. NY Acad. Sci
． , 663, 48-62, 1992).

By “fragment” of a polypeptide sequence is meant a polypeptide sequence that is shorter than its reference sequence but retains essentially the same biological function or activity as the reference polypeptide.

A “fragment” of a polynucleotide sequence refers to a polynucleotide sequence that is shorter than the reference sequence of SEQ ID NO: 1.

“Variant” means a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide but retains its basic properties. A typical variant of a polynucleotide differs in base sequence from the reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not change the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in 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. Generally, the alterations are such that the sequences of the reference polypeptide and the variant are very similar overall and are limited in many regions to be identical. The variant and the reference polypeptide are 1 in the amino acid sequence.
It may differ by any combination of one or more substitutions, insertions, deletions. Amino acid residues that are substituted or inserted may or may not be encoded by the genetic code. Typical conservative substitutions include Gly, Al
a; Val, Ile, Leu; Asp, Glu; Asn, Gln; Se
r, Thr; Lys, Arg; as well as Phe and Tyr. A variant of a polynucleotide or polypeptide may be a naturally occurring variant, such as an allele, or a variant not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides can also be made by mutagenesis techniques or direct synthesis. Also included as variants are one or more post-translational modifications, such as glycosylation, phosphorylation,
It is a polypeptide having methylation, ADP-ribosylation and the like. Embodiments include N-terminal amino acid methylation, serine and threonine phosphorylation, and C-terminal glycine modification.

“Allele” means one of two or more alternative forms of a gene occurring at a given locus in the genome.

“Polymorphism” refers to the nucleotide sequence (at a given position in the genome within a population (
And, when relevant, mutations in the encoded polypeptide sequence.

“Single nucleotide polymorphism” (SNP) refers to the occurrence of base variability at a single base position in the genome within a population. SNPs can occur within genes or within intergenic regions of the genome. SNP is an allele specific amplification (ASA)
Can also be assayed. This method requires at least 3 primers. The common primer is used in reverse complement to the polymorphism being assayed. This common primer is spaced 50-1500 base pairs from the polymorphic base. The other two (or more) primers are the last 3 '
The bases are equal to each other, except that the bases shift to fit one of the two (or more) alleles of the polymorphism. Two (or more) PCR reactions are then performed on the sample DNA, each with one of the common and allele-specific primers.

As used herein, a “splice variant” is initially the same genomic DN.
It refers to a cDNA molecule transcribed from the A sequence but produced from an RNA molecule that has undergone alternative RNA splicing. Alternative RNA splicing is
Multiple m's, in which the original RNA transcript occurs when spliced, generally to remove introns, each of which may encode a different amino acid sequence
Causes the production of RNA molecules. The term splice variant refers to the aforementioned cDNA
It also means the protein encoded by the molecule.

“Identity” reflects the relationship between two or more polypeptide sequences, or two or more polynucleotide sequences, and is determined by comparing the sequences. In general, identity refers to an exact base-to-base or amino acid-to-amino acid correspondence of two polynucleotides or two polypeptides, respectively, over the entire length of the sequences being contrasted.

"% Identity" -For sequences that do not have an exact match, "% Identity" can be determined. Generally, the two sequences to be contrasted are aligned to give the maximum correlation between the sequences. This may include inserting "gaps" in one or both sequences to increase the degree of alignment. The percent identity can also be determined over the entire length of each contrasting sequence (so-called global alignment), which is particularly suitable for sequences of identical or very similar length, or , Can also be done for shorter, defined lengths (so-called local alignment), which is better suited for arrays of different length.

“Similarity” is a further, more sophisticated measure of the relationship between two polypeptide sequences. In general, "similarity" refers to evolutionary evolution in the absence of exact correspondence, as well as exact correspondence between each pair of corresponding residues of the sequences being compared (as well as identity). By reason, it means a comparison between the amino acids of the two polypeptide chains, residue by residue, also taking into account whether one residue is likely to replace the other. This likelihood has an associated "score", based on which 2
The "% similarity" of two sequences can be determined.

Methods to compare the identities and similarities of two or more sequences are well known in the art. That is, for example, Wisconsin Sequence A
programs available in analysis package, version 9.1 (
Devereux J et al. , Nucleic Acids Res
． , 12, 387-395, 1984, Genetics Compu.
ter Group, available from Madison, Wis., USA), for example, the programs BESTFIT and GAP to determine the percent identity between two polynucleotides and the percent identity and similarity between two polypeptide sequences. Can be used. BESTFIT is for Smith and Wa
terman (J. Mol. Biol., 147, 195-197,
1981, Advances in Applied Mathematics
s, 2, 482-489, 1981) "local homology" algorithm is used to find one region where the similarity between two sequences is highest. B
ESTFIT is better suited for the comparison of two polynucleotide or two polypeptide sequences that are dissimilar in length, such that the short sequence represents a portion of the long sequence. I assume. On the other hand, GAP is Neddl
eman and Wunsch (J. Mol. Biol., 48, 443.
The alignment of the two sequences is performed according to the algorithm of -453, 1970) to find the "maximum similarity". GAP is about the same length,
It is also more suitable for comparison of sequences where alignment is expected over its entire length. The "gap weight" and "length weight" parameters used in each program are preferably 50 and 3 for polynucleotide sequences and 12 and 4 for polypeptide sequences, respectively. Percent identity and similarity are preferably determined when the two contrasting sequences are optimally aligned.

Other programs for determining identity and / or similarity between sequences are also known in the art, eg, the BLAST program family (
Altschul S F et al. , J. Mol. Biol. ，
215, 403-410, 1990, Altschul S Fet.
al. , Nucleic Acids Res. , 25: 389-3402.
, 1997, National Center for Biotecno
logy Information (NCBI), Bethesda, MD, USA, available at www. ncbi. nlm. nih. gov's NCBI home page) and FASTA (Pearson W.
R. , Methods in Enzymology, 183, 63-9.
9, 1990; Pearson W. R. and Lipman D.D.
J. , Proc. Nat. Acad. Sci. USA, 85, 2
444-2448, 1988, Wisconsin Sequence A.
(available as part of the analysis package).

BLOSUM62 Amino Acid Substitution Matrix (Henikoff San
d Henikoff J G, Proc. Nat. Acad Sci.
USA, 89, 10915-10919, 1992), for comparison of polypeptide sequences, including the case of previously translating a base sequence into an amino acid sequence, before comparison.
It is preferable to use.

The program BESTFIT is preferably used to determine the percent identity of a polynucleotide or polypeptide sequence to be evaluated to a reference polynucleotide or polypeptide sequence, as described hereinabove in the program. Set the parameters to their default values to optimally align the sequence to be evaluated with the reference sequence.

“Identity Index” is a measure of sequence relatedness that can be used to compare a candidate sequence (polynucleotide or polypeptide) to a reference sequence. That is, compared to the reference polynucleotide sequence, for example, candidate polynucleotide sequences having an identity index of 0.95 average 5 per 100 bases of the reference sequence.
It is in agreement with the reference sequence except that it has up to 4 differences. Such differences are selected from the group consisting of at least one nucleotide deletion, substitutions including transitions and transversions, or insertions. These differences may occur at the 5'or 3'ends of the reference polynucleotide sequence, or at any position between these end sites, either individually in the nucleotides of the reference sequence, or It may be interspersed within one or more contiguous groups within the reference array. In other words, in order to obtain a polynucleotide sequence having an identity index of 0.95 as compared with the reference polynucleotide sequence, as described above in this specification, an average of 5 nucleotides is calculated for every 100 bases of the reference sequence. Deletions, substitutions or insertions up to, or
Any combination thereof may be made. Other values of the identity index, for example 0.96,
The same applies mutatis mutandis for 0.97, 0.98 and 0.99.

Similarly, for a polypeptide, compared to a reference polypeptide sequence, for example, a candidate polypeptide sequence having an identity index of 0.95 has an average of 5 amino acids per 100 amino acids of the reference sequence. Consistent with the reference sequence, except with differences. Such differences are selected from the group consisting of at least one amino acid deletion, substitutions including conservative and non-conservative substitutions, or insertions. These differences may occur at amino- or carboxy-terminal sites of the reference polypeptide sequence, or at any position between these terminal sites, and may occur within the reference sequence amino acids, individually, or within the reference sequence. , Interspersed in one or more consecutive groups. In other words, in order to obtain a polypeptide sequence having an identity index of 0.95 as compared to the reference polypeptide sequence, as described above in the present specification, an average value is calculated for every 100 amino acids of the reference sequence. Up to 5 deletions, substitutions or insertions, or any combination thereof may be made. Other values of the identity index, eg 0
． Make the necessary changes for 96, 0.97, 0.98 and 0.99,
The same applies.

The relationship between the number of nucleobase or amino acid differences and the identity index can be represented by the formula: n _a ≦ x _a − (x _a · I) In the formula, n _a is the number of differences in the nucleic acid bases or amino acids, and x _a is the respective bases or amino acids in SEQ ID NO: 1 or SEQ ID NO: 2. Is the total number, I is the identity index, is the symbol for the multiplication operator, and if the product of x _a and I is not an integer, rounds it to the nearest integer before subtracting it from x _a To do.

“Homolog” is a general term used in the art to refer to a polynucleotide or polypeptide sequence that has a high degree of sequence relatedness to a reference sequence. Such relatedness can be quantified by determining the identity and / or similarity between two sequences, as defined herein. The terms "ortholog" and "paralog" are included in this general term. “Ortholog” means a polynucleotide or polypeptide that is functionally equivalent to a polynucleotide or polypeptide of another species. "Paralogue" means a polynucleotide or polypeptide that is functionally similar within the same species.

“Fusion protein” means a protein encoded by two unrelated fused genes or fragments thereof. Examples are U.S. Pat. No. 5,541,087, U.S. Pat. No. 5,726,044, European Patent No. 574395, European Patent No. 493418.
And EP 0232262. Fc-ANIC-B
In the case of P-2, in order to improve the pharmacokinetic properties of such a fusion protein when used for therapy and also to generate dimeric Fc-ANIC-BP-2, Fc-
It is advantageous to use the immunoglobulin Fc region as part of the fusion protein to effect functional expression of ANIC-BP-2. Fc-ANIC-BP-2 DN
The A construct comprises, in the 5'to 3'direction, a secretion cassette, ie, a signal sequence that triggers release from mammalian cells, DNA encoding an immunoglobulin Fc region fragment as a fusion partner, and Fc-ANIC-BP-. DN coding 2
Including A. In some applications, it does not affect the rest of the fusion protein but is functional Fc
It is desirable to mutate the side to alter its intrinsic functional properties (complement binding, Fc receptor binding) or to allow the Fc portion to be completely removed after expression.

All publications and publications, including but not limited to patents and patent applications, cited herein are referred to by the individual publication or publication as if fully cited. Are specifically incorporated herein by reference in their entirety, and if specifically and individually indicated, are incorporated herein by reference in their entirety. Any patent application to which this application claims priority is incorporated herein by reference in its entirety in the manner previously described for publications and literature. Further Examples Example 1 Traumatic Brain Injury Model The identification of genes up or down regulated in response to traumatic brain injury (TBI) was tested in rats using the lateral flood method. Male S
Moderate TB concentrated in the right parietal cortex in prague-Dawley rats
I was induced by the lateral flood percussion method. Five days after TBI, the rats were sacrificed and the excised brain tissue was analyzed by differential display. Protein upregulation was confirmed by RT-PCR in the cerebellum of traumatized brain.

A control group of rats was anesthetized and the temporalis muscle contracted, but no craniotomy was performed. 5
After survival for one day, all rats were anesthetized, sacrificed, their brains were removed and frozen in liquid nitrogen.

A second group of TBI rats was used for histochemical and immunohistochemical staining. One week after TBI, the rats were anesthetized and perfused with saline followed by 3% paraformaldehyde. The brain was cut into 30 μm coronal sections on a freezing microtome.

Example 2 Immunohistochemistry Cerebellar sections were labeled with a monoclonal antibody against the calcium binding protein. Immune complexes were visualized using the avidin-biotin / DAB method. As a positive control, Calbindin-D (28 kD) was used as a reliable marker for cerebellar Purkinje cells.

Example 3 In-Situ Hybridization Oligodeoxynucleotides (sense, antisense) selected from SEQ ID NO: 1 were designed according to standard methods well known to those skilled in the art. These probes were used for in situ hybridization in rat brain tissue sections according to methods known in the art. Autoradiograph is Kodak BioMax-F
After exposure on the ilm for 5 days, it was developed.

Example 4 RNA Isolation from TBI Rats mRNA differential display was developed as a method to identify and analyze alterations in gene expression at mRNA levels in any eukaryotic cell (Liang. and Pardee, Science 257
, 967, 1992). In the present invention, in order to gain more insight into the molecular effects of CNS damage, we used this method to test genes up or down regulated in response to traumatic brain injury (den).
Daas et al. Meeting of the America
n Neuroscience Society Washington D.N.
C. , USA; 1998). The animal model of the traumatic brain injury is called the lateral flood percussion model and is performed as follows. Male Sprague
In e-Dawley rats, the lateral flood percussion method induced moderate traumatic brain injury concentrated in the right parietal cortex of the brain. Control rats were anesthetized and contracted in the temporal muscle, but no craniotomy was performed. After survival for 5 days, the rats were anesthetized, sacrificed, their brains removed and frozen in liquid nitrogen. Whole brain was homogenized and total RNA was isolated (Sambrook et al., 1989). Differential Display The obtained RNA was analyzed by RNA differential display. mR
Reverse transcription of NA was performed with an oligo dT primer (downstream primer; 13 bases long) with two additional bases in all possible combinations, so the reaction was at the beginning of the poly (A) tail. Be stopped. Amplification of cDNA was performed using the same 3'primer and an optional 5'primer with a second 10 base length (upstream primer). Amplification products were analyzed by non-denaturing 10% polyacrylamide gel (Amersham).
(Pharmacia Biotech, Germany). DNA was visualized by silver staining. After staining, the gel was dried at room temperature for 1 hour (Fig. 1). The differentially controlled bands were excised from the gel. DNA was eluted, reamplified, and pCR2.
1 vector (Invitrogen, USA). The subcloned fragment was subjected to the Sanger method (Sanger F. et al., P.
NAS USA, 74, 5463-5467) and the sequences were compared to a genomic database. Confirmation of the obtained gene fragment was confirmed by reverse transcription PCR (R
T-PCR). To confirm the specifically expressed rat Mo25, specific titers of 19 bases and 21 bases were used to detect Titan.
One-tube RT-PCR system (Boehringer Mannhei
Sequences were analyzed by RT-PCR using m., Germany). For RT-PCR,
1 μg of total RNA from control and TBI rats was used. Further gene confirmation was carried out by the dot blotting method using a probe derived from rat Mo25 and human Mo25.
It was performed using Northern blot analysis with the probe from. Reverse Transcription PCR (RT-PCR) To confirm the specifically expressed stroke-inducing calcium-binding protein, the Titan one-tube RT-PCR system (Boehringer Mannheim, Boehringer Mannheim, Germany)
The sequence was analyzed by RT-PCR using. For the RT-PCR, 1 μg of total RNA from control and TBI animals was used. Real-time PCR The distribution of FKBP-25 in the human brain was examined by the real-time TaqMan PCR method on the ABI Prism 7700 Sequence Detection System (PE, Applied Biosystems, Germany). By this method, the absolute concentration of mRNA can be measured with high sensitivity. A 25- and 29-base long specific primer and a 32-base long TaqMan probe (reporter dye: FAM / quencher dye: TAMRA) were designed. Ca ²⁺ binding Proteins separated by SDS-PAGE were blotted onto nitrocellulose membranes for radiolabeled Ca ²⁺ binding by Maruyama, K. et al. e
t al. (J. Biochem. 95: 511-519, 1984). After incubation, excess isotope was washed and the membrane was washed with Kodak XOMAT. Exposed to TM film for appropriate time. The band was developed at the location of the bound protein. The presence of the binding protein was confirmed by adding the antibody using an antibody specific for the binding protein by Western blotting well known in the art.

[Sequence list]

[Brief description of drawings]

Brief Description of the Drawings Figure 1 shows a multi-tissue Northern blot duplicate experiment of the human ANIC-PB-2 gene with immobilized RNA from rat heart, brain, spleen, lung, liver, skeletal muscle (SKM), kidney and testis. , Very high levels of labeling occur in the testis, heart and brain;
Liver, skeletal muscle and kidney produced moderate levels of labeling, and spleen and lungs produced minimal levels of labeling.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 1/19 C12N 1/21 4H045 1/21 C12P 21/02 C 5/10 C12Q 1/02 C12P 21 / 02 1/68 A C12Q 1/02 G01N 33/15 Z 1/68 33/50 Z G01N 33/15 33/53 Z 33/50 C12N 15/00 A 33/53 5/00 A (71) Applicant Frankfurter Str. 250, D-64293 Darmstadt, Federal Republish of Germany (72) Inventor Denders, Isaac Germany 64407 Krunbach Schlastraße 76 (72) Inventor Duker, Klaus Germany 64291 Darmstadt Ette 5 F-term (reference) 2G045 AA35 BB24 BB48 DA13 DA36 FB03 FB09 4B024 AA01 AA11 BA44 BA80 CA04 CA09 CA11 DA01 DA02 DA05 DA11 EA01 EA02 EA03 EA04 FA02 GA11 HA01 HA03 HA12 4B063 QA01 QA18 QA19 QQ01 QQ42 QQ52 QR08 QR33 QR42 QR55 QR59 QR62 QR74 QR80 QS05 QS25 QS36 QX02 4B064 AG01 CA01 CA19 CC24 DA01 DA13 4B065 AA01X AA57X AA87X AA93Y AB01 AB02 BA01 BA08 CA24 CA25 CA44 CA46 4H045 AA10 AA11 AA20 AA30 BA10 BA41 CA40 DA00 DA76 FA72 EA50 FA50

Claims (11)

[Claims] 1. An (a) isolated polypeptide encoded by a polynucleotide comprising the sequence of SEQ ID NO: 1; (b) at least 95% identity to the polypeptide sequence of SEQ ID NO: 2. An isolated polypeptide comprising a polypeptide sequence having: c) an isolated polypeptide having at least 95% identity to the polypeptide sequence of SEQ ID NO: 2; d) a polypeptide of SEQ ID NO: 2 And (e) an isolated polypeptide selected from one of the group consisting of fragments and variants of the polypeptides of (a)-(d). 2. The isolated polypeptide of claim 1, which comprises the polypeptide sequence of SEQ ID NO: 2. 3. The isolated polypeptide of claim 1, which is the polypeptide sequence of SEQ ID NO: 2. 4. An isolated polynucleotide comprising (a) a polynucleotide sequence having at least 95% identity to the polynucleotide sequence of SEQ ID NO: 1; (b) the polynucleotide of SEQ ID NO: 1. (C) a polynucleotide sequence encoding a polypeptide sequence having at least 95% identity to the polypeptide sequence of SEQ ID NO: 2. An isolated polynucleotide comprising: (d) an isolated polynucleotide having a polynucleotide sequence encoding a polypeptide sequence having at least 95% identity to the polypeptide sequence of SEQ ID NO: 2; (e) Has the sequence of SEQ ID NO: 1 or at least 15 bases under stringent hybridization conditions An isolated polynucleotide comprising a base sequence having a length of at least 100 bases, which is obtained by screening a library with a labeled probe having a fragment of: (f) (a) to (e) A polynucleotide which is an RNA equivalent; or a polynucleotide sequence which is complementary to said isolated polynucleotide, and variants and fragments of said polynucleotide or which are complementary to said polynucleotide over its entire length An isolated polynucleotide selected from one of the group consisting of: 5. (a) an isolated polynucleotide comprising the polynucleotide of SEQ ID NO: 1; (b) an isolated polynucleotide of SEQ ID NO: 1; (c) encoding the polypeptide of SEQ ID NO: 2. The isolated polynucleotide of claim 4, selected from the group consisting of: an isolated polynucleotide comprising a polynucleotide sequence; and (d) an isolated polynucleotide encoding the polypeptide of SEQ ID NO: 2. 6. An expression system comprising a polynucleotide capable of producing the polypeptide of claim 1 when said expression vector is present in a compatible host cell. 7. A recombinant host cell comprising the expression vector of claim 6 or a membrane thereof, which expresses the polypeptide of claim 1. 8. A method of producing the polypeptide of claim 1, wherein the host cell of claim 7 is cultured under conditions sufficient to produce the polypeptide,
Then, a method comprising the step of recovering the polypeptide from the medium. 9. A fusion protein comprising an immunoglobulin Fc region and any one of the polypeptides according to claim 1. 10. An immunospecific antibody against the polypeptide according to any one of claims 1 to 3. 11. A screening method for identifying a compound that stimulates or inhibits the function or concentration of the polypeptide according to claim 1, which comprises (a) the polypeptide (or the polypeptide is expressed). Cell or membrane)
Alternatively, quantitatively or qualitatively measuring or detecting the binding of the candidate compound to the fusion protein by a label directly or indirectly linked to the candidate compound; (b) in the presence of a labeled competitor. Measuring the competition of binding of a candidate compound to said polypeptide (or cells or membranes expressing said polypeptide) or its fusion protein; (c) compatible with cells or cell membranes expressing said polypeptide Detecting whether the candidate compound produces a signal caused by activation or inhibition of the polypeptide using a detection system according to claim 1; (d) the candidate compound containing the polypeptide of claim 1. To form a mixture, measure the activity of the polypeptide in the mixture, and determine the activity of the mixture as a candidate. Or (e) detecting the effect of the candidate compound on the production of mRNA encoding said polypeptide or said polypeptide in the cell, for example using an ELISA assay. A method selected from the group consisting of: and (f) producing the compound according to biotechnology or chemical standard procedures.