JP2003503024A - Head injury-induced cytoplasmic calcium-binding protein - Google Patents

Abstract

  (57) [Summary] ANIC-BP polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods of using ANIC-BP polypeptides and polynucleotides in diagnostic assays.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to human-acute nerve-inducible calcium-binding protein (ANIC-BP), as well as the corresponding polynucleotide encoding this protein. The invention also provides expression vectors, host cells as well as antibodies. In addition, the present invention provides a method for producing the protein, and a method for treating or preventing a disease associated with the expression of the protein. The invention also 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 applicable treatment to date. 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 attacks are
In Western countries, it is a predominant disease in young people. 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. Using the technique of arterial reperfusion,
Further research is on its way. Although early clinical trials with polymorphonuclear leukocyte-dependent endothelial adhesion receptor antagonists are being completed, approaches must still be created. However, both approaches, which target only a single stage in the ischemic cascade, produce only limited benefits. Therefore,
Future therapies will 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. However, 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 Ca ²⁺ level regulation 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 report 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, 1288-1292, 1990). However, cultured hippocampal neurons expressing high levels of calbindin-D28K have been reported to be selectively resistant to glutamate-induced toxicity (Baimbrid).
ge et al., TINS, 15, 303-8, 1992). Similarly, hippocampal neurons expressing high levels of calretinin, excitotoxins glutamate, NMDA,
Resistant to toxic doses of kainate and quisqualate (Winsky et al
., Novel Calcium-Binding Proteins, 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. Neuron.
l., 111, 293-301, 1991) and Huntington's disease (Kiyama et al., Brain R)
es., 526, 303-07, 1990), it was reported to be selectively vulnerable.
Calbindin-D28K-expressing neurons in the substantia nigra are not selectively vulnerable during Parkinson's disease (Yamada 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 specific hippocampal cell types (To.
rtosa Et al., Neurosci., 1, 33-43, 1993), but another study suggested that hippocampal interneurons that express parvalbumin are selectively vulnerable during Alzheimer's disease. (Brady et al., Neurosci., 80, 1113-25, 199 7).

[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. , Deficient in function (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, retroviral infection of motor neurons with calbindin-D28k has been shown to have a neuroprotective effect on IgG-induced toxicity from patients with amyotrophic lateral sclerosis (Ho. et al., PNAS, 9
3, 6796-801, 1996), and transfection of calbindin-D28k has been shown to protect PC12 cells from serum withdrawal, glutamate exposure, and toxicity due to the neurotoxin MPP + (McMahon et al. ., Molec. Brai
n 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. Therefore, there is considerable evidence that CaBP confers resistance as well as vulnerability in the CNS injury process, but the mechanisms involved and the regulation of the response still have to be elucidated.

A gene family containing a functionally unidentified gene (MO25) was recently isolated from a mouse-derived cDNA library (Miyamoto et al., Mol. Reprod. D
ev., 34, 1-7, 1993). This library contains R isolated from early mouse embryos.
Built from NA. From the predicted amino acid sequence of Mo25, it was revealed that the MO25 gene has structural homology with the Ca ²⁺ -binding protein and lacks the transmembrane domain. Suggest that it may be involved in the development of. 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 the Mo25 cDNA is similar to that of the mouse Mo25 homolog 6
It had a homology of 9.3%. The homologue in Saccharomyces cerevisiae was encoded in an open reading frame near the calcineurin B subunit gene. Recently, another gene is Aspergillus hy
was isolated from the mA mutant (Karos et al., Mol. Gen Genet., 260, 510-521.
, 1999), corresponding to homologues in 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 is a major tool for high-throughput DNA sequencing technology, as well as various bioinformatics tools for identifying gene sequences of potential interest from many molecular biology databases currently available. Depends on. 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 provides ANIC-BPs, and in particular ANIC-BP polypeptides and ANs.
It relates to an IC-BP polynucleotide, a recombinant thereof, and a method for producing them. Such polypeptides and polynucleotides include, but are not limited to, stroke and acute head trauma, multiple sclerosis and spinal cord injury, hereinafter referred to as a "disease of the invention", There is interest in the treatment of diseases. In a further aspect, the invention relates to methods of identifying agonists as well as antagonists (eg, inhibitors) using the materials provided by the invention, and the compounds identified are associated with an ANIC-BP imbalance. It relates to a method of treating a condition. In yet another aspect, the present invention provides an improper ANI.
It relates to a diagnostic assay for detecting a disease associated with the activity or concentration of C-BP.

[0011]     (Explanation of the invention)   In a first aspect, the present invention relates to ANIC-BP 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% relative to 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 variants of the polypeptides of (a)-(f). Is included.

The polypeptides of the 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 are hereinafter referred to as “ANIC-BP biological activity” or “ANIC-BP activity”. The polypeptides of the invention preferably exhibit at least one ANIC-BP biological activity.

The polypeptides of the present invention also include variants of the aforementioned polypeptides, including 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 that mediate the biological activity of ANIC-BP, including those with similar or improved activity, or with reduced undesirable activity. Also, in animals, especially humans,
Fragments that are antigenic or immunogenic are also preferred.

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 present invention relates to ANIC-BP polynucleotides. 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. is there. 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., DNA
cell Biol., 15, 505-09, 1996) and Mo25 (Karos et al., Mol. Gen.
Genet., 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 Hym
^* Related to other proteins of the calcium-binding protein family that have homology and / or structural similarities to the 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 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., Molecular Cloning: A Labora.
tory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY (1989)). 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 provided by Gentz et al., Proc. Natl. Acad. Sci. USA (1989) 86: 821. Hexa-histidine peptide or HA tag, as described in US Pat.
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 for 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 present invention, including homologues from a species other than human, will preferably have a sequence of SEQ ID NO: 1 or a sequence thereof of at least 15 bases under stringent hybridization conditions. It may be obtained by a method comprising screening a library with a labeled probe having a fragment and isolating a full-length cDNA as well as a genomic clone containing the polynucleotide sequence. Such hybridization techniques are well known to those of ordinary skill in the art. Suitable, 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 denatured and cleaved salmon sperm DNA 20
Incubation overnight at 42 ° C. in a solution containing microgram / ml; followed by washing the filters in 0.1 × SSC at about 65 ° C. Accordingly, the invention is obtained by screening a library under stringent hybridization conditions, preferably with at least 15 bases, with 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 isolated cDNA sequences are often incomplete and that 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 (eg Frohman et al., Proc. Nat. Acad.
Sci. USA 85, 8998-9002, 1988). Recent improvements, exemplified for example by the Marathon ™ method (Clontech Laboratories Inc.), have significantly simplified the search for longer cDNAs. Marath
In the on ™ method, cDNA is prepared from mRNA extracted from selected tissues and a “adapter” sequence is ligated to each end. Nucleic acid amplification (PCR) is then performed using the gene-specific as well as adapter-specific combination of oligonucleotide primers to amplify the "missing"5'end of the DNA. Then, a “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). PCR using a gene-specific primer that anneals to the 5 ′ side of the sequence.
The reaction is repeated. The product of this reaction is then analyzed by DNA sequencing and the product ligated directly to the existing cDNA to give the complete sequence, or
Utilizing the new sequence information to design the 5'primer, a separate full-length P
Depending on whether CR is performed, full-length cDNA can be constructed.

The recombinant polypeptides of the present 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 Molecular Biology (1986) and Sambr.
It can be introduced into host cells by methods described in many standard laboratory manuals, such as ook et al. (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 Streptococcus, Staphylococcus, Escherichia coli, Streptomyces and Bacillus subtilis cells, fungal cells such as yeast cells and Aspergillus cells, Drosophila S2 and Hasmonyoto Sf9 cells. Insect cells, animal cells such as CHO, COS, 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 expression systems such as bacterial plasmid derived, bacteriophage derived, transposon derived, yeast episome derived, insert factor derived, yeast chromosomal factor derived, baculovirus, SV40. Vectors derived from viruses such as papovavirus, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and retrovirus, and cosmid and phagemid, and those derived from plasmids and bacteriophage genetic factors, etc. Vectors derived from those combinations can be used. The expression system may include control regions that induce and at the same time regulate expression. Generally, any system or vector capable of maintaining, increasing, or expressing a polynucleotide can be used for production of the polypeptide in a host. The appropriate polynucleotide sequence may also be inserted into the expression system by any of a variety of well-known and conventional techniques, such as those set forth in Sambrook et al. (Supra), supra. 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. Detection of a mutant form of the 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 spatial expression 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 cDNA
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 nucleotide 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 by direct DNA sequencing (eg, Myers et al., Science (1
985) 230: 1242)). Sequence changes at specific sites can be linked to nuclease protection assays, such as RNase and S1 protection,
Alternatively, a chemical cleavage method (Cotton et al., Proc. Nat.
l. Acad. Sci. USA (1985) 85: 4397-440.
1)).

Arrays of oligonucleotide probes comprising ANIC-BP polynucleotide sequences 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 .; Chee
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
A subject's susceptibility to the disease of the present invention can also be used for diagnosing or determining. Decreased or elevated expression can be achieved, for example, by nucleic acid amplification, eg PC
It can be measured at the RNA level using any of the methods well known in the art for quantifying polynucleotides, such as R, RT-PCR, RNase protection, Northern blotting, and other hybridization methods. it can. 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 assays include
Included are 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 disease of the present 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 genetic map data. Such data is, for example, V.I. McKusick's Mendelian Inheritance in Man (Jo
hns Hopkins University Welch Medical
(Available online from Library). 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). Genome sequence (gene fragment, etc.)
The exact position on the human chromosome for is the radiation hybrid (RH) mapping (Walter, M., Spillet, D., Thomas, P., W.
eissenbach, J .; , And Goodfellow, P .; , (1
994) A method for constructing a radiation hybrid map of the whole genome (A method)
for constructing radiation hybrid m
aps of whole genomes), Nature Geneti
cs 7, 22-28). Some RH panels, such as the GeneBridge4 RH panel (Hum. Mol.
Genet. 1996 Mar; 5 (3): 339-46 Radiation hybrid map of human genome.
f the human genome); Gyapay G, Schmi
tt K, Fizzames C, Jones H, Vega-Czarn
y N, Spillett D, Muselet D, Prud'Hom
me JF, Dib C, Affray C, Morissette
J, Weissenbach J, Goodfellow PN)
Available from search Genetics (Huntsville, Ala., USA). This panel was used to determine the chromosomal location of the gene to determine the RH
Using a primer designed from the gene of interest on DNA, 93 times of P
Carry out CR. Each of these DNAs has a hamster background (
Human / hamster hybrid cell lines) containing random human genomic fragments. These PCRs give 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 is http
/// www. genome. wi. mit. Do it with edu.

The polynucleotide sequences of the present invention are also useful tools for studying tissue expression. Such studies can determine the expression pattern of a polynucleotide 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. And
The techniques used are well known in the art and include cDNA microarray hybridization (Schena et al., Science, 27).
0, 467-470, 1995 and Shalon et al. , Ge
nome Res. , 6, 639-645, 1996), and in situ hybridization method for clones arranged on a lattice, and PCR.
Including nucleotide amplification methods such as. The preferred method is Perkin Elmer
The TAQMAN ™ method available from The results of these studies can also give an indication of the normal function of the polypeptide in that organism. In addition, the normal expression pattern of mRNA is expressed by the expression pattern of RNA encoded by a variant form of the same gene (eg, one that has a mutated potential or an altered ability to encode a polypeptide or a regulatory mutation). The contrast test with can also provide useful information regarding the role of the polypeptide of the present invention, or the role of its inappropriate expression during disease. Such inappropriate expression may be characteristic in time, space or simply quantitatively. The polypeptides of the present invention are expressed in human brain.

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 producing 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.

An antibody generated against the polypeptide of the present invention is prepared by administering a fragment containing the polypeptide or epitope, or cells to an animal, preferably a non-human animal, using a conventional protocol. Can be obtained by For preparation of monoclonal antibodies, any technique which provides antibodies produced by subculturing cell lines can be used. Examples include 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 a.
l. , Monoclonal Antibodies and Cancer
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 invention provides a method for inducing an immune response in a mammal which protects the animal from the disease, whether or not the disease is already chronic in the individual. In order to do so, a method comprising inoculating said mammal with an antibody and / or a polypeptide of the invention, which is suitable for generating a T cell immune response comprising, for example, cytokine producing T cells or cytotoxic T cells. Regarding An immune response in a mammal encodes the polypeptide in vivo and in vivo for the purpose of inducing such an immune response to produce an antibody in order to protect said animal from the diseases of the present invention. It can also be induced by a method comprising delivery of a polypeptide of the invention via a vector that directs expression of nucleotides. One means of administering the vector is by promoting it into the desired cells as a coating on particles or the like. Such nucleic acid vector may be composed of DNA, RNA, modified nucleic acid, or DNA / RNA hybrid. For use in vaccines, the polypeptide or nucleic acid vector is usually a vaccine formulation (composition).
Will be provided as. The formulation may further comprise a suitable carrier. Polypeptides can also be broken down in the stomach, so parenteral (eg, subcutaneous,
It is preferably administered intramuscularly, intravenously or intradermally). 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 adjuvant 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 diseases according to the invention 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 are various raw materials,
For example, it can be identified from 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; structural or functional mimetics thereof (Coligan et al., Current P
rotocols in Immunology 1 (2): Chapter 5 (19
91)) or a small molecule.

The screening method simply measures the binding of a candidate compound to the polypeptide, a cell or membrane having the polypeptide, or a fusion protein thereof by a label directly or indirectly bound to the candidate compound. You may. 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 may use a detection system suitable for cells having the polypeptide to examine whether the candidate compound produces a signal generated by activation or inhibition of the polypeptide. Inhibitors of activation are generally assayed in the presence of a known agonist, and the effect of the presence of the candidate compound on activation by the agonist is observed. Furthermore, in the screening method, in order to form a mixture, a step of mixing the candidate compound with a solution containing the polypeptide of the present invention, a step of measuring ANIC-BP activity in the mixture, and an ANIC-BP of the mixture.
Comparing the activity to a control mixture containing no candidate compound.

The polypeptides of the present invention can also be used in conventional low performance screening methods and in high throughput screening (HTS) formats. Such an HTS format is not only well-established use of 96-well, and more recently 384-well microtiter plates, but also Schule
k et al. , Anal. Biochem. , 246, 20-29
, (1997), including newly 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 Method) The polynucleotide, polypeptide and antibody against the polypeptide of the present invention are used to form a screening method for detecting the effect of an added compound on the production of mRNA and polypeptide in cells. You can also For example, monoclonal assays as well as polyclonal antibodies can be used to construct an ELISA assay for measuring 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 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,
The polypeptide is labeled with a radioisotope (eg, ¹²⁵ I), chemically modified (eg, biotinylated), or fused to a peptide sequence suitable for detection or purification, and a putative receptor source (Cells, cell membranes, cell supernatants, tissue extracts,
Incubate with body fluids), ligand binding as well as cross-linking assays. Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy.
These screening methods can also be used to identify any agonists and antagonists of the polypeptide that compete with the binding of the polypeptide to its receptor. Standard methods for performing such assays are well understood in the art.

Examples of antagonists of the polypeptides of the present invention include antibodies or, in some cases, oligonucleotides or proteins which are intimately associated with the polypeptide's ligands, substrates, receptors, enzymes, etc. 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.

The screening method can also include the use of transgenic technology and the ANIC-BP gene. Techniques for constructing transgenic animals are well established. For example, the ANIC-BP gene can be used for microinjection of a fertilized oocyte into a male nucleus, introduction of a retrovirus into an embryo before or after transplantation, or an embryonic stem cell genetically modified by electroporation or the like. It can be introduced by injection 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 transgenic animal technology also provides a whole animal expression-cloning system in which the introduced gene is expressed to provide large quantities of the polypeptide of the invention.

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 facilitate understanding of certain terms frequently used above.

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 in said organism, and that organism is It 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 together by peptide bonds or modified peptide bonds, ie, peptide isosteres. "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 genetically encoded amino acids. A “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 reference books and more detailed research papers, 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 attachment of flavins, covalent attachment of heme moieties, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, phosphotidyl. Inositol covalent bond, cross linking, cyclization, disulfide bond formation, demethylation, covalent cross link formation, cystine formation, pyroglutamate formation, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic treatment, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation, and other transfer RNA-mediated amino acid addition to proteins , And ubiquitination (eg, protein Quality - the nature of the structure and molecular, Second Edition (Prote
ins-Structure and Molecular Property
es, 2nd Ed. ), T.S. E. Creighton, W.M. H. Fr
eeman and Company, New York, 1993; Post-translational covalent modification of proteins (Post-translational Covalen).
t Modification of Proteins), B.I. C. Jo
hson Ed. W., Academic Press, New York, New York. , Post-translational protein modification: Future perspectives and expectations (Post-tra
nstaltional Protein Modifications: Per
Species and Prospects), 1-12; Seift
er et al. "Analysis of protein modification and non-protein cofactors (Analy
sis for protein modifications and no
nprotein cofactors, "Meth. Enzymol.
, 182, 626-646, 1990, and Rattan et a.
l. , "Protein Synthesis: Post-translational modification and aging (Protein Synt
hesis: Post-translational Modifatio
ns and Aging) ", Ann. NY Acad. Sci. , 6
63, 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.

A “variant” differs from a reference polynucleotide or polypeptide, but
A polynucleotide or polypeptide that retains its basic properties. A typical variant of a polynucleotide differs in nucleotide 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. In general, the alterations are such that the sequences of the reference polypeptide and variant are very similar overall and are limited in many regions to be identical. Variants and reference polypeptides may differ in amino acid sequence by one or more substitutions, insertions, deletions in any combination. Amino acid residues that are substituted or inserted may or may not be encoded by the genetic code. Typical conservative substitutions include Gly, Ala; Val, Ile, Leu; Asp, Glu.
Asn, Gln; Ser, Thr; Lys, Arg; and 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 polypeptides having one or more post-translational modifications, such as glycosylation, phosphorylation, 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 that occur at a given locus in the genome.

“Polymorphism” means a variation in the nucleotide sequence (and, in the context, the encoded polypeptide sequence) at a given position in the genome within a population.

“Single nucleotide polymorphism” (SNP) means the occurrence of nucleotide 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 (A
SA) can also be used for the assay. This method requires at least 3 primers. The common primer is used in reverse complement to the polymorphism being assayed. This common primer is from 50 to 1500 from polymorphic bases.
The base pairs are separated. The other two (or more) primers, except that the last 3'base is changed such that it matches one of the two (or more) alleles of the polymorphism, Equal to each other. Then 2 with sample DNA
Multiple (or more) PCR reactions are performed, each with one of the common and allele-specific primers.

As used herein, a “splice variant” is initially the same genomic D
RNA transcribed from the NA sequence but subjected to alternative RNA splicing
A cDNA molecule produced from a molecule. Alternative RNA splicing results in the production of multiple mRNA molecules, which occur when the original RNA transcript undergoes splicing, generally to remove introns, each of which may encode a different amino acid sequence. . The term splice variant refers to the above-mentioned cDNA.
It also means the protein encoded by the A 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 correspondence, "% 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 of the sequences being contrasted (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 sequences of different length.

“Similarity” is an even 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 on a residue-by-residue basis, taking into account whether one residue is likely to replace the other. This likelihood has an associated "score" on which the "% similarity" of the two sequences can be determined.

Methods of comparing the identities and similarities of two or more sequences are well known in the art. That is, for example, Wisconsin Sequence An
programs available in the Alysis Package, version 9.1 (D
evereux J et al. , Nucleic Acids Res.
, 12, 387-395, 1984, Genetics Comput.
er Group, available from Madison, Wisconsin, USA), for example:
The programs BESTFIT and GAP can be used to determine the% identity between two polynucleotides, and the% identity and similarity between two polypeptide sequences. BESTFIT is Smith and Wat
erman (J. Mol. Biol., 147, 195-197, 1)
981, Advances in Applied Mathematics
, 2, 482-489, 1981) “local homology” algorithm is used to find one region where the similarity between two sequences is highest. BE
STFIT is better suited for the comparison of two polynucleotide or two polypeptide sequences that are dissimilar in length, with the program saying that short sequences represent a portion of a longer sequence. I assume. On the other hand, GAP is Neddle
man and Wunsch (J. Mol. Biol., 48, 443-.
453, 1970) to align the two sequences so as 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. The percent identity and similarity are preferably determined when the two sequences to be contrasted are optimally aligned.

Other programs for determining identity and / or similarity between sequences are also known in the art, eg, the BLAST family of programs (Al.
tschul S F et al. , J. Mol. Biol. , 21
5, 403-410, 1990, Altschul S F et al.
． , Nucleic Acids Res. , 25: 389-3402,
1997, National Center for Biotechnolo
gy Information (NCBI), available from Bethesda, MD, USA, www. ncbi. nlm. nih. gov's NCBI home page) and FASTA (Pearson WR, M)
methods in Enzymology, 183, 63-99, 19
90; Pearson WR and Lipman DJ, Proc.
Nat. Acad. Sci. USA, 85, 2444-2448,
1988, Wisconsin Sequence Analysis P
available as part of the package).

BLOSUM62 Amino Acid Substitution Matrix (Henikoff S and
Henikoff J G, Proc. Nat. Acad Sci.
USA, 89, 10915-10919, 1992) is preferably used for comparison of polypeptide sequences, including the case of previously translating a nucleotide sequence into an amino acid sequence before comparison.

The program BESTFIT is preferably used to determine the percent identity of the evaluated polynucleotide or polypeptide sequence with respect to the reference polynucleotide or polypeptide sequence, as previously described herein.
Set the program parameters to their default values to optimally align the evaluated and reference sequences.

“Identity index” is a measure of sequence relatedness that can be used to compare a candidate sequence (polynucleotide or polypeptide) with a reference sequence.
That is, as compared with the reference polynucleotide sequence, for example, the identity index 0
． The candidate polynucleotide sequence having 95 is consistent with the reference sequence, except that it has an average of up to 5 differences per 100 bases of the reference sequence. 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, individually within the nucleotides of the reference sequence, Alternatively, they may be interspersed within one or more contiguous groups within the reference sequence. 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 the present specification, the average value is calculated for every 100 bases of the reference sequence. Up to 5 deletions, substitutions or insertions, or any combination thereof may be made. The same applies, mutatis mutandis, for other values of the identity index, for example 0.96, 0.97, 0.98 and 0.99.

Similarly, for a polypeptide, as compared to the reference polypeptide sequence,
For example, a candidate polypeptide sequence having an identity index of 0.95 matches the reference sequence, except that it has an average of 5 differences per 100 amino acids of the reference sequence. 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 the amino- or carboxy-terminal sites of the reference polypeptide sequence, or at any position between these terminal sites, individually within the amino acids of the reference sequence, or It may be interspersed in one or more consecutive groups within the reference array. 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 this specification, for every 100 amino acids of the reference sequence, An average of up to 5 deletions, substitutions or insertions, or any combination thereof may be made. Other values of the identity index, such as 0.96, 0.97, 0.98 and 0.99
The same applies, mutatis mutandis.

The relationship between the number of base or amino acid differences and the identity index can be expressed by the following formula: n _a ≦ x _a − (x _a · I) In the above formula, n _a is the number of differences of bases or amino acids, and x _a is of the respective bases or amino acids of 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, in order to improve the pharmacokinetic properties of such fusion protein when used for therapy and also to generate dimeric Fc-ANIC-BP, Fc-ANIC
-Immunoglobulin Fc as part of the fusion protein to effect functional expression of BP
It is advantageous to use regions. The Fc-ANIC-BP DNA construct is 5 '
In the 3'to 3'direction, it comprises a secretion cassette, ie, a signal sequence that triggers excretion from mammalian cells, DNA encoding an immunoglobulin Fc region fragment as a fusion partner, and Fc-ANIC-BP encoding DNA. In some applications, it does not affect the rest of the fusion protein, but mutates the functional Fc side to alter its inherent functional properties (complement binding, Fc receptor binding), or
It is desirable 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 hereby incorporated by reference as if each individual publication or publication is fully incorporated by reference. When incorporated into the specification, it is hereby incorporated by reference in its entirety as if specifically and individually indicated. 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 Exemplification 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 Sp
Moderate TBI concentrated in the right parietal cortex of the rague-Dawley rat 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 with a freezing microtome.

Example 2 Immunohistochemistry Cerebellar sections were labeled with a monoclonal antibody for calcium binding proteins. 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 selected from SEQ ID NO: 1 (sense, antisense) were designed according to standard methods apparent 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 on Kodak BioMax-Film
Visualization was performed after 5 days of exposure.

Example 4 RNA Isolation from TBI Rats An mRNA differential display was developed as a method for identifying and analyzing mutant gene expression at the mRNA level in any eukaryotic cell (Liang and Pardee, Science 257, 967,
1992). In the present invention, we used this method to test up- or down-regulated genes in response to traumatic brain injury in order to gain more insight into the molecular effects of CNS damage (den Daas). e
t al. Meeting of the American Neuro
science Society Washington D.A. C. , USA;
1998). The animal model for traumatic brain injury is called the lateral flood percussion model and is performed as follows. Lateral flood percussion in male Sprague-Dawley rats induced moderate traumatic brain injury focused in the right parietal cortex of the brain. Control rats were anesthetized to contract the temporal muscle,
No craniotomy was performed. After survival for 5 days, the rats were anesthetized, sacrificed, their brains removed and frozen in liquid nitrogen. The whole brain was homogenized and total RNA was isolated (S
ambrook et al. , 1989).

Differential Display The obtained RNA was analyzed by RNA differential display. mR
Oligo d with reverse transcription of NA with two additional bases in all possible combinations
The T primer (downstream primer; 13 bases long) was used and therefore the reaction was immobilized at the beginning of the poly (A) tail. Amplification of cDNA was performed using the same 3'primer and an optional 5'primer with a second 10 base length (upstream primer). The amplified product was subjected to non-denaturing 10% polyacrylamide gel (Amersham P
(Harmcia 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, pCR2.1
Subcloned into vector (Invitrogen, USA). The subcloned fragment was sequenced by the Sanger method (Sanger F. et.
al. , PNAS USA, 74, 5463-5467), sequences were compared to a genomic database. Confirmation of the obtained gene fragment was confirmed by reverse transcription PCR (RT
-PCR). To confirm the specifically expressed rat Mo25, the sequence was analyzed by RT-PCR using the Titan one-tube RT-PCR system (Boehringer Mannheim, Germany) with specific 19- and 21-base length primers. . For RT-PCR, 1 μg of total RNA from control group and TBI rat was used. For further gene confirmation, rat Mo
It was carried out using dot blotting with a probe from 25 and Northern blot analysis with a probe from human Mo25.

Reverse Transcription PCR (RT-PCR) To confirm the specifically expressed stroke-inducing calcium binding protein, Titan One Tube R with specific 19- and 21-base primers.
Sequences were analyzed by RT-PCR using the T-PCR system (Boehringer Mannheim, Germany). Control group and TBI for RT-PCR
1 μg of total RNA from rat was used.

Real-time PCR The distribution of ANIC-BP in rat brain after head injury was analyzed by real-time Taq.
ABI Prism 7700 Sequence Detection System (PE, Ap
probed by Biosystems, Germany). By this method,
The absolute concentration of NA can be measured with high sensitivity. Specific primers having a length of 25 and 29 bases and a TaqMan probe having a length of 32 bases (reporter dye: FAM /
Quencher dye: TAMRA) was designed.

Ca ²⁺ Binding Proteins separated by SDS-PAGE were blotted onto nitrocellulose membranes and radiolabeled Ca ²⁺ binding was performed by Maruyama, K. et al. et
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]

FIG. 1 shows a representative mRNA contrasting display gel with specifically regulated bands in the contralateral cerebellar hemisphere on the injured side. Arrows indicate specifically expressed bands.

FIG. 2 shows a 270 base pair gene fragment of rat Mo25 in the cerebellum after TBI,
FIG. 5 shows RT-PCR for 5, 10, 15, 20, 25 and 30 cycles, respectively. Note the strong expression in the L2 lane at 20, 25 and 30 cycles. L2: cDNA of non-injured side of TBI rat; R2: cDNA of injured side of TBI rat
.

FIG. 3: Radioactive ³² P-labeled 2 of rat Mo25 using rat cerebellum mRNA after TBI.
FIG. 6 shows dot blot analysis with 70 base pair gene fragment. Six individual rats were tested, three were TBI treated and three were sham treated.

FIG. 4: Multi-tissue Northern blot (Clontech) hybridized to 8 rat tissues with a radioactive ³² P-labeled 270 base pair gene fragment of rat Mo25.
Laboratories Inc, Palo Alto, CA, USA.

FIG. 5 shows in-system hybridization of rat brain slices. Sense and antisense probes specific for SICCBP and Mo25 were used. Myelinated nerve cords (callosal line, optic line, middle olfactory line, precerebellar commissure) when antisense probe is used
The strong signal shown by was observed.

FIG. 6: Real-time Ta of ANIC-BP in rats 7 days after traumatic brain injury.
It is a figure which shows qMan PCR amplification. The uninjured cortex side, the injured cortex side, and the cerebellum were compared by the brain tissue of sham-treated rat brains and rats sacrificed 7 days after the experiment. Error bars are the standard error of the mean (SEM).

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 1/15 C12N 1/19 4H045 1/19 1/21 1/21 C12P 21/02 C 5/10 C12Q 1/02 C12P 21/02 G01N 33/15 Z C12Q 1/02 33/50 Z G01N 33/15 33/53 D 33/50 C12N 15/00 ZNAA 33/53 5/00 A (81) Designated country EP ( AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, B , KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE , DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ , TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (71) Applicant Frankfurter Str. 250, D-64293 Darmstadt, Federal Republish of Germany (72) Inventor Denders, Itzak Germany Day-64407 Frenquisch-Krumbach Schlästraße 76 (72) Inventor Fischer, Fiora Federal Republic of Germany 55-122 Than der Ally 80 (72) Inventor Seyfried, Christoph Germany Day – 64 342 Seeheim Martildenstraße 6 (72) Inventor von Melchner, Raleigh Germany Day – 64380 Rossdorf Heinrich Heine-Strasse 6 ． F-Term (reference) 2G045 AA40 DA36 FB03 4B024 AA01 AA11 CA04 CA09 HA14 4B063 QA01 QA18 QR48 QS03 QS34 4B064 AG01 CA19 CC24 DA01 DA13 4B065 AB01 BA02 CA24 CA44 CA46 4H045 AA10 AA11 A50 CA23 CAA 4 A0

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; and (d) SEQ ID NO: 2 An isolated polypeptide selected from the group consisting of: (e) (a) to (d) fragments and variants of the polypeptide. 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. (a) An isolated polynucleotide comprising 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) at least 15 Using a labeled probe having the nucleotide of SEQ ID NO: 1 or a fragment thereof. It is obtained by screening a library under stringent hybridization conditions, at least 100
An isolated polynucleotide comprising a nucleotide sequence of bases; (f) a polynucleotide which is an RNA equivalent of the polynucleotides of (a) to (e); or a polynucleotide sequence complementary to said isolated polynucleotide, and An isolated polynucleotide selected from the group consisting of variants and fragments of the above-mentioned polynucleotide over its entire length, or a polynucleotide that is complementary to the above-mentioned polynucleotide. 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, which is 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 such an expression vector is present in a compatible host cell. 7. A recombinant host cell or a membrane thereof comprising the expression vector according to claim 6, which expresses the polypeptide according to claim 1. 8. A method for producing the polypeptide of claim 1, wherein the host cell of claim 7 is cultured under conditions sufficient for production of the polypeptide,
Then, a method comprising a step of recovering the polypeptide from the medium. 9. A fusion protein comprising an immunoglobulin Fc region and any one of the polypeptides of 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 promotes or inhibits the function or concentration of the polypeptide of claim 1, which comprises (a) the polypeptide (or expresses the polypeptide). Cell or membrane) or its fusion protein, quantitatively or qualitatively, by quantitatively or qualitatively measuring or detecting the binding of the candidate compound directly or indirectly with a label associated with the candidate compound; (b) a label The competition of the binding of the candidate compound to the polypeptide (or cells or membranes expressing the polypeptide) or its fusion protein in the presence of the selected competitor; (c) the poly CIGNA produced by activation or inhibition of the polypeptide using a detection system suitable for cells expressing the peptide or cell membrane Le
Testing whether or not the candidate compound causes; (d) mixing the candidate compound with a solution containing the polypeptide of claim 1 to form a mixture, wherein the mixture of the polypeptide in the mixture is Measuring the activity and comparing the activity of said mixture to a control mixture which does not contain said candidate compound; or (e) for the intracellular production of mRNA encoding said polypeptide or said polypeptide, Detecting the effect of the candidate compound using, for example, an ELISA assay; and (f) producing the compound according to a conventional biotechnology or chemical method. A screening method comprising.