JP2002253252A - Method for assaying cytotoxic activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new method for assaying a cytotoxic activity, and a method for screening a compound affecting cytotoxic activity using the method. SOLUTION: This fused polypeptide (53 amino acid length) comprises a part of a protein transportation domain located (corresponding to 47-57 residue among 86 amino acid residues) at the C end side of TAT protein of HIV-1 virus and Aβ protein of 42 amino acid length connected to the C end side of the protein of the part.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for measuring cytotoxic activity on a nerve cell by introducing a fusion polypeptide into the nerve cell, and a method for screening for a compound that promotes or suppresses the cytotoxic activity. More specifically, the present invention relates to a method for measuring a neuronal cytotoxic activity using an amyloid β synthetic polypeptide known as one of the causes of Alzheimer's disease, or a method for screening a compound that affects the activity.

[0002]

BACKGROUND OF THE INVENTION Alzheimer's dementia is characterized pathologically by the formation of senile plaques, neurofibrillary tangles and loss of nerves. Amyloid β protein (Aβ) is a protein consisting of about 40 amino acid residues and is a major component of senile plaques. Aβ accumulation in the brain is the earliest pathological change, Aβ is toxic to nerve cells, and the gene responsible for familial Alzheimer's disease (am
It is considered that Aβ protein is one of the causes of Alzheimer-type dementia, because a large amount of Aβ is accumulated in the brain of transgenic mice (yloid precursor protein: APP).

On the other hand, in APP transgenic mice, a large amount of Aβ accumulation similar to senile plaques is observed extracellularly, but the interpretation of apparent neuronal death accompanying Aβ accumulation is inconsistent (Irrizarry, MC , Soria
no, F., McNamara, M., etal., J. Neurosci., 17, 705
3-7059 (1997): Irizarry, MC, McNamara, M., Fedorc
hak, K., et al., J. Neuropathol. Exp. Neurol., 56,
965-973 (1997): Calhoun, ME, Wiederhold, KH, Ab
ramowski, D. et al., Nature, 395, 755-756 (199
8)).

[0004] On the other hand, in a transgenic mouse related to familial Alzheimer's disease (PS1), A
Large accumulation of extracellular Aβ as seen in the PP transgenic mouse brain is not observed, but intracellular Aβ accumulation and associated neuronal death have been observed
(Chui, DH, Tanahashi, H., Ozawa, K. et al., Natur
e Med., 5, 560-564 (1999)). Aβ is produced intracellularly from precursor protein, but it has been reported that Aβ sequence 1-42 accumulates in neurons prior to senile plaque formation and neurofibrillary tangles even in Alzheimer's patient brain. (Gouras, GK, Tsai, J., Naslund, J., et al., Amer
ican J. Pathol., 156, 15-20 (2000)). These results indicate that Aβ in cells is independent of senile plaque formation.
In particular, it has been suggested that accumulation of Aβ sequence 1-42 may promote neuronal cell death.

It has been reported that cell overexpression of APP or the C-terminal fragment of APP containing Aβ causes cell death (International Publication No .: APP695: WO2000 / 14204).
Among the peptides suitable for cytotoxicity of amyloid production, there is a peptide consisting of 10 amino acid sequences including a GAIL sequence (JP-A-11-71393), but Aβ itself accumulates in cells and promotes cell injury. There are no reports of doing so.

Therefore, an experimental system that accumulates Aβ in cells and promotes cytotoxicity is useful for analyzing the mechanism of new cytotoxicity caused by intracellular Aβ and for screening candidate compounds that affect cytotoxicity. Conceivable.

On the other hand, it is known that the TAT protein of the HIV-1 virus, when added to cells, passes through the cell membrane and enters the cells and the nucleus. Full length and fragmentation T
It has been reported that when other proteins and oligopeptides are bound to the AT protein, these proteins and oligopeptides are transported into cells together with the TAT protein while maintaining their original biological functions ( Fawell, S., See
ry, J., Daikh, Y., et al., Proc. Natl. Acad. Sci.
USA, 91, 664-668 (1994): Nagahara, H., Vocero-Akba
ni, AM, Snyder, EL et al., Nature Med., 4, 144
9-1452 (1998)).

[0008]

DISCLOSURE OF THE INVENTION The present invention relates to a method for accumulating Aβ polypeptide associated with Alzheimer's disease in nerve cells, measuring the neuronal cytotoxic activity of the polypeptide, and the cytotoxic activity of the polypeptide. It is an object of the present invention to provide a method for screening a compound that gives a change.

[0009]

To provide a method for measuring a change in cytotoxic activity due to accumulation of Aβ protein in cells as described above, the present inventors have conducted intensive studies and as a result, found that the TAT peptide sequence (Ie, amino acid sequences 47-57) and a polypeptide consisting of a synthetic sequence of a polypeptide consisting of the amino acid sequences 1-42 of Aβ protein are accumulated in cells by contact with the cells. We succeeded in promoting high cytotoxicity and completed the invention here.

That is, the present inventors linked 53 polypeptides consisting of the A42 protein 1-42 amino acid sequence to the C-terminal side of an 11 amino acid peptide which is a part of the protein transport domain of the TAT protein. Has been shown to cause a high degree of neuronal damage by its uptake and accumulation in neurons upon contact with the neurons, indicating that the polypeptide has cytotoxic activity. It has been confirmed that a method for measurement and a method for screening candidate compounds that change the cytotoxic activity thereof can be provided.

According to the present invention, there is further provided a method for measuring cytotoxic activity on a nerve cell by introducing a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 into the nerve cell. The present invention provides a method for measuring cytotoxic activity, which is a primary cultured cell of the cortical nerve, and / or the cytotoxic activity is measured by morphological changes and / or the number of living or dead cells.

Further, according to the present invention, there is provided a method for screening for a compound which promotes or suppresses a nerve cell-damaging activity by introducing a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 into a nerve cell. A method for screening a therapeutic / prophylactic agent for Alzheimer's by introducing a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 into a nerve cell, and a method for screening a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 into a nerve cell. A method for screening for a compound that promotes or suppresses neuronal cytotoxicity by transfection, comprising: (a) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or Contacting the test sample together, (b) cytotoxicity of said nerve cells in both of (a) And (c) selecting a compound that promotes or suppresses the cytotoxicity of the nerve cell. Further, in the screening method, the cytotoxic activity is measured by morphological changes and / or the number of living or dead cells,
A method is provided for screening for a compound that promotes or suppresses the neuronal cytotoxic activity, wherein the neuron is a primary cultured cell of cortical nerve of the brain.

Further, according to the present invention, there is provided a screening method wherein the compound which inhibits the neuronal cytotoxicity due to the introduction of the polypeptide into nerve cells is an antibody binding to the polypeptide or an antibody fragment thereof. Further, in the above antibody, it is possible to provide the above antibody having a binding ability to the Aβ sequence 1-42 amino acid sequence of the polypeptide.

According to the present invention, there is further provided a synthetic polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, a nucleic acid molecule encoding the polypeptide, and a set comprising the amino acid sequence represented by SEQ ID NO: 1 encoded by the nucleic acid molecule. Provided is a recombinant polypeptide, an antibody that binds to the synthetic polypeptide or the recombinant synthetic polypeptide or an antibody fragment thereof, wherein the antibody has an ability to bind to the Aβ sequence 1-42 amino acid sequence of the polypeptide. can do.

Further, according to the present invention, a method for measuring cytotoxic activity on nerve cells by introducing a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 into nerve cells, and a method for measuring changes in the cytotoxic activity thereof. The polypeptide used in the method for screening a given candidate compound can be provided which is expressed by a nucleic acid molecule encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1.

According to the present invention, there is provided a compound which suppresses nerve cell injury obtained by a screening method for a candidate compound which changes the cytotoxic activity of a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 on nerve cells. Or a therapeutic / prophylactic agent for Alzheimer's disease comprising the compound as an active ingredient.

Further, according to the present invention, a gene having a variation in gene expression is detected in a nerve cell by culturing the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 and a nerve cell in a suitable medium. Method and a gene or gene expression product detected by the method for detecting the gene, a nerve cell containing the gene or gene expression product, a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 alone, or the polypeptide Gene and gene expression by a method comprising the steps of: contacting a test sample with a test sample, detecting cytotoxicity of both neurons, and selecting a compound that promotes or suppresses neuronal cytotoxicity. Measuring the neuronal cytotoxic activity of the product and determining the agonist / antagonist for the activity of the gene or gene expression product. A method for detecting a gonist is provided.

Hereinafter, the abbreviations for amino acids, (poly) peptides, base sequences, nucleic acids and the like in the present specification are shown as follows:
The IUPAC and IUB regulations, "Guidelines for the preparation of specifications containing base sequences or amino acid sequences" (Japan Patent Office), and commonly used symbols in the art shall be followed.

A specific example of the polypeptide used in the method of the present invention or in the method of screening for a candidate compound that changes cytotoxic activity in a nerve cell is SEQ ID NO: 1 shown in Example 1 described later. Is a polypeptide consisting of the amino acid sequence represented by

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION The method for measuring a neuronal cytotoxic activity by the polypeptide of the present invention and the method for screening a candidate compound that changes the cytotoxic activity thereof will be described in detail below.

That is, as described above, a method for measuring a neuronal cytotoxic activity by a polypeptide of the present invention and a method for obtaining / synthesizing a polypeptide for use in a method for screening a candidate compound that changes the cytotoxic activity, The method for expressing and obtaining a recombinant using a nucleic acid molecule encoding a peptide will be described in detail, and a method for measuring cytotoxic activity using the obtained synthetic polypeptide or recombinant polypeptide will be exemplified.

First, the polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 used in the measurement method of the present invention is HIV-1.
Fusion of a portion of the protein transport domain located on the C-terminal side of the TAT protein of the virus (corresponding to residues 47-57 of 86 amino acids) and a 42-amino acid long Aβ protein connected to its C-terminal side It is a polypeptide (53 amino acids long).

The Aβ protein of 42 amino acids in the polypeptide of the present invention has 1 to 5 (preferably 1 to 3) amino acids deleted as long as it exhibits the following cytotoxic activity. It may be substituted, inserted or added. For example, 11 derived from the TAT protein of the HIV-1 virus
A spacer sequence may be included between the peptide having an amino acid length and the polypeptide derived from the Aβ protein. For example, a spacer sequence such as 3 to 5 glycine residues may be included between both polypeptides. Preferably,
It is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1.

The polypeptide of the present invention can be produced by a general chemical synthesis method in accordance with the amino acid sequence, and includes a conventional liquid phase method and a solid phase peptide synthesis method. . More specifically, such a peptide synthesis method is based on amino acid sequence information, a stepwise erosion method in which each amino acid is sequentially linked one by one to extend the chain, and a fragment consisting of several amino acids is synthesized in advance, and then And a fragment condensation method in which each fragment is subjected to a coupling reaction. The synthesis of the synthetic polypeptide of the present invention may be performed by any of these methods.

The condensation method employed in the above peptide synthesis also includes
Various methods can be followed. Specific examples thereof include azide method, mixed acid anhydride method, DCC method, active ester method, redox method, DPPA (diphenylphosphoryl azide) method, and DCC + additives (1-hydroxybenzotriazole, N-hydroxysuccinamide). , N-hydroxy-5-norbornene-2,3-dicarboximide), the Woodward method and the like. The solvent that can be used in each of these methods can be appropriately selected from general solvents that are well known to be used in this kind of peptide condensation reaction. Examples include dimethylformamide (DMF), dimethylsulfoxide (DMSO),
Hexaphosphoramide, dioxane, tetrahydrofuran (THF), ethyl acetate and the like, and a mixed solvent thereof can be mentioned. In the above peptide synthesis reaction,
Carboxyl groups in amino acids and peptides not involved in the reaction are generally esterified to form lower alkyl esters such as methyl ester, ethyl ester, and tertiary butyl ester such as benzyl ester and P-
It can be protected as methoxybenzyl ester, P-nitrobenzyl ester aralkyl ester and the like.
In addition, the hydroxyl group of an amino acid having a functional group in a side chain, for example, Tyr may be protected with an acetyl group, a benzyl group, a benzyloxycarbonyl group, a tertiary butyl group, or the like, but such protection is not necessarily performed. . Further, for example, a guanidino group of Arg includes a nitro group, a tosyl group, a 2-methoxybenzenesulfonyl group, a methicylene-2-sulfonyl group,
It can be protected by a suitable protecting group such as a benzyloxycarbonyl group, an isobornyloxycarbonyl group and an adamantyloxycarbonyl group. The deprotection reaction of the protecting group in the amino acid and peptide having the above protecting group and the finally obtained synthetic polypeptide represented by SEQ ID NO: 1 used in the measurement method of the present invention can also be performed by a commonly used method such as contacting. It can be carried out according to a reduction method, a method using liquid ammonia / sodium, hydrogen fluoride, hydrogen bromide, hydrogen chloride, trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid or the like.

The thus-obtained polypeptide of the present invention can be obtained by a conventional method, for example, using an ion exchange resin,
Purification can be appropriately performed according to a method widely used in the field of peptide chemistry such as partition chromatography, gel chromatography, affinity chromatography, high performance liquid chromatography (HPLC), and countercurrent distribution method.

The polypeptide of the present invention can be prepared, for example, by synthesizing a polypeptide or a DNA nucleic acid molecule encoding the amino acid sequence of the polypeptide set forth in SEQ ID NO: 1 and then introducing it into an appropriate expression vector, followed by in a host cell. It can also be obtained by genetic engineering techniques obtained by expression.

More specifically, first, for example, SEQ ID NO:
An oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 encoding the amino acid sequence represented by No. 1 is synthesized. Examples of the method include chemical synthesis means such as the phosphate triester method and the phosphate amidite method [J. Am. Chem. Soc., 89, 4801 (19
67); id. 91, 3350 (1969); Science, 150, 178 (196
8); Tetrahedron Lett., 22, 1859 (1981); 24, 245
(1983)] and methods for combining them.
More specifically, the synthesis of DNA can be performed by a chemical synthesis using a phosphoramidite method or a triester method, or can be performed on a commercially available automatic oligonucleotide synthesizer. The double-stranded fragment synthesizes a complementary strand,
It can also be obtained from chemically synthesized single-stranded products by annealing the strands together under appropriate conditions, or adding a complementary strand using a DNA polymerase with appropriate primer sequences.

As described above, a specific embodiment of the DNA molecule of the present invention is a DNA molecule having the DNA sequence shown in SEQ ID NO: 2. This DNA molecule sequence
1 shows an example of one combination of codons indicating each amino acid residue in the amino acid sequence shown in SEQ ID NO: 1. Thus, a DNA molecule is a DNA having such a specific DNA sequence.
Not only the molecule but also any combination is possible as long as an arbitrary codon is combined with each amino acid residue, has a selected base sequence, and encodes the amino acid sequence shown in SEQ ID NO: 1.

In the amino acid sequence represented by SEQ ID NO: 1, 1 to 5 (preferably 1 to 3) amino acids are deleted, substituted, inserted or deleted as long as they exhibit the following cytotoxic activity. It may be a DNA encoding the added amino acid sequence. Preferably, SEQ ID NO: 2
Is a DNA molecule consisting of a base sequence represented by

The selection of codons can be performed according to a conventional method,
For example, the codon usage of the host to be used can be considered [Ncleic Acids Res., 9, 43 (1981)].

When obtaining DNA molecules, if the amount of DNA obtained above is small, the PCR method [Scie
nce, 230, 1350 (1985)].

The primer used when employing the PCR method can be appropriately set based on the sequence information of the DNA of the nucleic acid molecule, and can be synthesized according to a conventional method.
In addition, the isolation and purification of the amplified DNA fragment can be performed according to a conventional method as described above, for example, by gel electrophoresis.

The nucleic acid molecule or DNA fragment of the present invention obtained as described above can be prepared by a conventional method, for example, the dideoxy method [Proc.
Acad. Sci., USA., 74, 5463 (1977)) and the Maxam-Gilbert method (Methods in Enzymology, 65, 499 (19
80)] and the base sequence can be conveniently determined using a commercially available sequencing kit or the like.

Next, the DNA product of the present invention obtained as described above is transformed into the recombinant product (expressed protein having the same amino acid sequence as the synthetic polypeptide) or the recombinant product by using ordinary genetic engineering techniques. Can be easily and stably produced in large quantities.

Accordingly, the present invention also encompasses the fusion Aβ synthetic polypeptide represented by SEQ ID NO: 1 obtained above, a DNA molecule encoding the amino acid sequence thereof, and a recombinant polypeptide expressed by the DNA molecule. Is what you do.

Further, according to the present invention, the recombinant product, such as a polypeptide such as a fusion Aβ polypeptide,
For producing the polypeptide, for example, the DNA of the present invention
The present invention also provides a vector containing the molecule, a host cell transformed with the vector, and a method for producing the polypeptide of the present invention by culturing the host cell.

The method for producing and expressing the above polypeptide is as follows.
It can be manufactured by referring to Ohno et al., "Protein Experiment Protocol 1 Functional Analysis, Cell Engineering Separate Volume, Experimental Protocol Series, 1997, Shujunsha".

The polypeptide of the present invention can be prepared by a conventional gene recombination technique [eg, Science, 224, 1431] based on the sequence information of the DNA molecule provided by the DNA molecule.
(1984); Biochem. Biophys. Res. Comm., 130, 692 (1
985); Proc. Natl. Acad. Sci., USA., 80, 5990 (198
3) etc.].

The production of the polypeptide is more particularly described
A recombinant DNA (expression vector) capable of expressing a DNA molecule encoding the desired polypeptide in a host cell is prepared, introduced into a host cell, transformed, and the transformant is cultured. This is done by harvesting from the resulting culture.

As the host cell, any of prokaryote and eukaryote can be used. For example, prokaryote hosts include a wide variety of commonly used hosts such as Escherichia coli and Bacillus subtilis. Escherichia coli, especially those contained in the Escherichia coli K12 strain can be exemplified. Eukaryotic host cells also include
Cells such as vertebrates and yeast are included, and the former includes, for example, COS cells [Cell, 23: 175 (198
1)] and Chinese hamster ovary cells and their dihydrofolate reductase-deficient strains (Proc. Natl. Acad. Sc.
i., USA., 77: 4216 (1980)]. As the latter, yeast cells of the genus Saccharomyces are preferably used. Of course, it is not limited to these.

When a prokaryotic cell is used as a host, a promoter and an SD (Shine and & Co.) are used upstream of the gene so that a DNA molecule can be expressed in the vector using a vector that can be replicated in the host cell. (Dargano) base sequence, and an expression plasmid to which an initiation codon (eg, ATG) necessary for initiation of protein synthesis is added can be suitably used. As the above vector, generally, a plasmid derived from Escherichia coli, for example, pBR322, pBR325, pUC1
2, pUC13 and the like are often used, but are not limited thereto, and various known vectors can be used.
Commercially available products of the above-mentioned vectors used in an expression system using Escherichia coli include, for example, pGEX-4T (Amersham Pharm
acia Biotech), pMAL-C2, pMAl-P2
(New England Biolabs), pET21, pET21 /
lacq (Invitrogen), pBAD / His (Invitr)
ogen).

When a vertebrate cell is used as a host, an expression vector usually includes a promoter located upstream of a DNA molecule to be expressed, a splice site of RNA,
Those having a polyadenylation site and a transcription termination sequence may be mentioned, which may further have an origin of replication if necessary. Examples of the expression vector include, for example, SV40
PSV2dhfr [Mol.
l. Biol., 1: 854 (1981)]. In addition to the above, various known commercial vectors can be used.
Commercially available products of such a vector used in an expression system using animal cells include, for example, pIND (Invitrogen),
vector for animal cells such as pcDNA3.1 / His (Invitrogen) or pFastBac HT (Gibc
iBRL), pAcGHLT (PharMingen), pA
c5 / V5-His, pMT / V5-His, pMT /
And insect cell vectors such as Bip / V5-his (all from Invitrogen).

Further, as a specific example of an expression vector when a yeast cell is used as a host, for example, pAM82 having a promoter for the acid phosphatase gene [Proc.
Natl. Acad. Sci., USA., 80: 1 (1983)]. Commercially available expression vectors for yeast cells include, for example, pP
ICZ (Invitrogen), pPICZα (Invitrogen
Company).

The promoter is not particularly limited. When Escherichia is used as a host, for example, tryptophan (trp) promoter, lpp promoter, lac
Promoters, recA promoters, PL / PR promoters and the like can be preferably used. When the host is a bacterium belonging to the genus Bacillus, SP01 promoter, SP02 promoter, penP promoter and the like are preferable. When a yeast is used as a host, the promoter may be, for example, pH
05 promoter, PGK promoter, GAP promoter, ADH promoter and the like can be suitably used.
When an animal cell is used as a host, preferred promoters include SV40-derived promoters, retrovirus promoters, metallothionein promoters,
Examples include a heat shock promoter, a cytomegalovirus promoter, and an SRα promoter.

The polynucleotide sequence whose coding sequence of the mature polypeptide assists the expression and secretion of the polypeptide from the host cell can be exemplified by a secretory sequence and a leader sequence. Marker sequence (hexahistidine tag, histidine tag), in the case of mammalian cells, hemagglutinin (H
A) ・ Tags can be exemplified.

The method for introducing a desired recombinant DNA (expression vector) into a host cell and the method for transforming the same with the host cell include the following.
There is no particular limitation, and various general methods can be employed.

The obtained transformant can be cultured according to a conventional method, and the desired polypeptide encoded by the DNA molecule designed as desired by the culture can be cultured inside, outside, or on the cell membrane of the transformant. Expressed, produced (accumulated, secreted).

As the medium to be used for the culture, various types of media commonly used depending on the host cells used can be appropriately selected and used, and the culture can be carried out under conditions suitable for the growth of the host cells.

The recombinant polypeptide of the present invention thus obtained can be subjected to various separation operations using its physical properties, chemical properties and the like [“Biochemical Data Book”, if desired.
II ”, pp. 1175-1259, 1st edition, 1st printing, June 23, 1980, published by Tokyo Chemical Co., Ltd .; Biochemistry, 25 (25), 8274
(1986); Eur. J. Biochem., 163, 313 (1987), etc.].

Specific examples of the method include ordinary reconstitution treatment, treatment with a protein precipitant (salting out method), centrifugation,
Osmotic shock method, sonication, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high-performance liquid chromatography (HPL
Examples thereof include various types of liquid chromatography such as C), dialysis methods, and combinations thereof. Particularly preferred methods include affinity chromatography using a column to which a specific antibody against the polypeptide of the present invention is bound. Can be.

In the present invention, an antibody that binds to the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 is produced by using the above-obtained synthetic polypeptide or recombinant polypeptide as an immunizing antigen. can do. It is preferable to use a carrier protein such as KLH in combination. More specifically, for example, a fused cell (hybridoma) of a mammalian plasma cell (immune cell) immunized with the above-mentioned immunizing antigen and a mammalian plasmacytoma cell is prepared, and the amino acid represented by SEQ ID NO: 1 is prepared therefrom. A clone producing a desired antibody recognizing the sequence is selected, and the clone can be produced by culture.

The mammal to be immunized with the immunizing antigen in the above method is not particularly limited, but is preferably selected in consideration of compatibility with the plasmacytoma cells used for cell fusion. Rats, rabbits and the like are advantageously used.

Immunization can be carried out by a general method, for example, by administering the above immunizing antigen to a mammal by intravenous, intradermal, subcutaneous, intraperitoneal injection or the like. More specifically,
The immunizing antigen is preferably administered to the test animal several times every 2 to 14 days, optionally in combination with a usual adjuvant, so that the total dose is about 100 to 500 μg / mouse. As an immunizing antigen, it is preferable to use spleen cells extracted about 3 days after the final administration.

Furthermore, various mammalian plasmacytoma cells as the other parent cells to be fused with the above-mentioned immunocytes include, for example, p3 (p3 / × 63-Ag).
8) [Nature, 256, 495-497 (1975)], p3-U1
[Current Topics in Microbiology and Immunology
, 81, 1-7 (1978)], NS-1 [Eur. Immuno
l., 6, 511-519 (1976)], MPC-11 [Cell, 8, 4
05-415 (1976)], SP2 / 0 [Nature, 276, 269-27
0 (1978)], FO [J. Immunol. Meth., 35, 1-21 (1
980)], × 63.6.5.3. [J. Immunol., 123,
1548-1550 (1979)] S194 [J. Exp. Med., 148,
313-323 (1978)], and R210 [Na
Nature, 277, 131-133 (1979)].

The fusion reaction between the above immune cells and plasmacytoma cells can be performed by a known method, for example, the method of Milstein et al.
d in Enzymology, Vol. 73, pp3 (1981)]. More specifically, the above-mentioned fusion reaction is carried out in a normal medium in the presence of a normal fusion promoter, for example, polyethylene glycol (PEG), Sendai virus (HVJ) or the like. An auxiliary agent such as dimethyl sulfoxide can be added as needed to increase the amount. The use ratio of immune cells to plasmacytoma cells is not different from the usual method. For example, it is common to use immune cells about 1 to 10 times as much as plasmacytoma cells. As the medium for the fusion reaction, various media usually used for the growth of plasmacytoma cells, for example, RP
An MI-1640 medium, a MEM medium, and other media generally used for this type of cell culture can be exemplified. Usually, it is preferable to remove a serum supplement such as fetal calf serum (FCS) from the medium. In the fusion, a predetermined amount of the above-mentioned immune cells and plasmacytoma cells are mixed well in the above-mentioned medium, and a PEG solution preliminarily heated to about 37 ° C. It is performed by adding and mixing at a concentration of 30 to 60% w / v%. Thereafter, a desired hybridoma is formed by successively adding an appropriate medium, centrifuging, and removing the supernatant.

The separation of the desired hybridoma obtained is
It is performed by culturing in a usual selection medium, for example, a HAT medium (a medium containing hypoxanthine, aminopterin and thymidine). The culture in the HAT medium may be performed for a period of time sufficient to kill cells (unfused cells and the like) other than the target hybridoma, usually several days to several weeks. The hybridoma thus obtained is subjected to a search for a target antibody and a single cloning by a usual limiting dilution method.

The search for the target antibody-producing strain is carried out, for example, by ELISA
Method A [Engvall, E., Meth. Enzymol., 70, 419-
439 (1980)], plaque method, spot method, agglutination reaction method, Ouchterlony method, radioimmunoassay (RIA) method, and other various methods generally used for detecting antibodies ["Hybridoma method and monoclonal method" Antibody ", published by R & D Planning Co., Ltd., No. 30-53
Page, March 5, 1982],
The immunizing antigen can be used for this search.

The hybridoma which produces the desired monoclonal antibody recognizing the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 can be subcultured in a usual medium, and can be cultured in liquid nitrogen for a long time. Can be saved.

The desired antibody can be collected from the hybridoma by a method of culturing the hybridoma according to a conventional method to obtain a culture supernatant, or by administering the hybridoma to a mammal compatible with the hybridoma and growing it. And the like. The former method is suitable for obtaining high-purity antibodies, and the latter method is suitable for mass production of antibodies.

The antibody obtained as described above can be further purified by usual means such as salting out, gel filtration, affinity chromatography and the like.

The monoclonal antibody of the present invention thus obtained has specific reactivity with the polypeptide having the amino acid sequence represented by SEQ ID NO: 1.

Further, according to the antibody of the present invention, the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 introduced into a nerve cell has a cytotoxic activity or by binding to the polypeptide, Antibodies of the type which have the activity of neutralizing or antagonizing the cytotoxic activity on neurons, especially on neuronal death such as morphological changes of neurons and increase of dead cells.
Such an antibody is suitable for specifically measuring the polypeptide. Antibodies of the type having such neutralizing or antagonistic activity, particularly those of the type recognizing a site involved in binding to Aβ sequence 1-42 on a polypeptide molecule having the amino acid sequence represented by SEQ ID NO: 1, It is suitable for application as a therapeutic agent for various diseases accompanied by an increase in the above-mentioned cytotoxic activity, particularly neuronal cell death, particularly Alzheimer's disease.

Further, the antibody of the present invention recognizes different sites of the polypeptide molecule having the amino acid sequence represented by SEQ ID NO: 1 and has no steric hindrance between the antibodies, and can simultaneously bind to the polypeptide molecule. Antibodies of possible types are also included, and such antibodies are extremely useful for use in immunoassays by, for example, the sandwich method.

In addition, the antibody of the present invention includes a type of antibody having particularly excellent reactivity in a liquid phase system or a solid phase system. They are highly preferred for application in liquid phase and solid phase immunoassays.

The measuring method and screening method of the present invention The measuring method of the cytotoxic activity of the present invention measures a neuronal cytotoxic activity by introducing a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 into a neuron. Is the way.

The nerve cells used in the method include, for example, cerebral nerve cells collected from mammals, such as mouse neuroepithelial cells, rat hippocampus primary cultured cells, mouse embryo cultured Purkinje cells, mouse dorsal root ganglia, and the like. An established cell line of brain neurons, such as a human glial cell line, human H
eLa cells, HUVEc cells, primitive human fibroblasts or lymphoblasts, human primitive mixed brain cells (neurons,
Stretched neurites by treating astrocytes and glia) with growth factors such as NGF (nerve growth and trophic factor) and IGF (insulin-like growth factor). More specifically, as the above-mentioned brain nerve cells, a culture solution obtained by extracting the tissue of a hippocampus of a mammal, for example, a rat, and culturing it in a complete medium is used. As those in which the established cell lines were treated with growth factors to extend neurites,
PC1 treated with NGF, FGF (fibroblast factor), EGF (epidermal growth factor), interleukin 6, etc.
2 cells, SH-SY5Y cells treated with IGF, NGF
Cells, NMB cells, NGP cells, SK treated with
-N-SH cells, LAN-1 cells, KA-9 cells, IM
R-32 cells, IMR-32 cells treated with 5-bromodeoxyuridine, NMB cells, NGP cells, and the like.

(1) Assay As an example of a method for assaying the cytotoxic activity of the present invention, rat hippocampal cells are used and a predetermined amount of a synthetic or recombinant polypeptide of the present invention is added to a culture medium of hippocampal cells. (Fusion polypeptide of amino acid sequence 47-57 of TAT protein and amino acid sequence 1-42 of amyloid β protein) under predetermined temperature conditions, and the behavior of neurons with time when contacted and / or Alternatively, by measuring the number of viable cells and comparing the number with the non-administration of the polypeptide, the desired neuronal cytotoxicity can be measured.

Hereinafter, a measurement method using, for example, rat hippocampus cells will be disclosed.

Hippocampal cells are prepared from the whole rat brain by a conventional method (for example, Example 2 (1)). To the obtained primary culture cells (0.1 to 1 × 10 ⁵ cells / cm ² ), an aqueous solution such as a medium containing the polypeptide of the present invention, a buffer solution, or water is added at an appropriate concentration (for example, 1 to 50 μM as a final concentration). ). As a control, the above aqueous solution not containing the polypeptide of the present invention is added. Thereafter, at about 30 to 40 ° C. in the presence of 5% CO ₂ for 1 to 72 hours, preferably 16 to 72 hours.
Culture and contact for ~ 48 hours.

At this time, the method described below can be used to measure the behavior of hippocampal cells and / or the number of viable cells over time.

(2) Screening Method The present invention provides a method for screening for a compound that promotes or suppresses the cytotoxic activity on nerve cells by introducing a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 into nerve cells. A method for screening a therapeutic / preventive agent for Alzheimer's by introducing a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 into a nerve cell, and introducing the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 into a nerve cell A method for screening for a compound that promotes or suppresses neuronal cytotoxicity by introducing a compound, comprising: (a) a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 alone or And contacting the test sample together, (b) both of (a) It is intended to provide a method comprising a step of detecting the cytotoxicity of the nerve cell, and a step (c) of selecting a compound that promotes or suppresses the cytotoxicity of the nerve cell. The screening method can be performed, for example, according to the following method.

The method for measuring the cytotoxic activity can be basically carried out according to the method described in the above (1). For example, only when a predetermined amount of a synthetic or recombinant polypeptide of the present invention is added to a culture solution of nerve cells obtained by the method described in the above (1) under a predetermined temperature condition, the compound is synthesized with a candidate compound. Or simultaneously with the recombinant polypeptide, or after adding the candidate compound, the polypeptide, or after adding the polypeptide, the candidate compound,
In the case of adding the polypeptide alone, detecting cytotoxicity by measuring the behavior of nerve cells and / or measuring the number of living or dead cells over time when added and contacted under predetermined temperature conditions, for example, By comparing with, it can be determined whether the candidate compound is a compound that promotes or suppresses neuronal cell injury,
Thus, compounds that promote or suppress the desired neuronal cytotoxic activity of the present invention can be screened.

In the above method (1) or (2), the observation of morphological changes of nerve cells, such as the behavior of hippocampal cells, can be performed by using the synthetic or recombinant polypeptide of the present invention, or the synthetic or recombinant polypeptide. Morphological changes of neurons in a sample before and after administration of a body polypeptide and a candidate compound can be determined by, for example, a phase contrast microscope (for example, magnification: 200 to 600).
It can be observed by a 2 × magnification or an immunohistochemical method using an antibody.

Alternatively, the measurement can be performed using the surviving cells after treatment with the polypeptide of the present invention as an index. For example, a normal cell should have a morphologically smooth contour and numerous neurites, while a degenerated cell exhibits an apoptotic-like morphology such as irregular shape, neurite degeneration, etc. It can be determined that the cell has died. In addition, the number of surviving cells and the number of dead cells before and after treatment of the polypeptide or the polypeptide and the candidate compound can be dyed with a dye, and the cytotoxic activity can be similarly measured by a phase contrast microscope or a fluorometer. As the dye for determining the number of living cells and the number of dead cells, for example, the staining of the number of living cells is the number of cells stained with Calcein AM, and the staining of the number of dead cells is propidium iodine (P
By counting the number of cells stained with Ropidium Iodide, respectively, it is possible to measure the cytotoxic activity. Further, it is possible to measure dead cells stained blue by the trypan blue dye exclusion test. Also, by staining the nucleus with Hoechst 33258 dye,
It is possible to measure dead cells with fragmented nuclei. Furthermore, it is also possible to measure the amount of dead cells by measuring the amount of lactate dehydrogenase released into the culture solution following cell death. On the other hand, by measuring formazan of tetrazolium salts (MTT, MTS, WST-1, WST-8, etc.) generated by living cells, it is possible to measure the number of remaining living cells.

In the above, candidate compounds used in the method of screening for a compound that promotes or suppresses neuronal cytotoxicity of the present invention include antibodies or antibody fragments, peptides, non-peptide compounds, synthetic compounds, fermentation products, Examples include cell extracts, plant extracts, animal tissue extracts, and plasma, and these compounds may be novel compounds or known compounds.

To carry out the measurement method or the screening method of the present invention, the polypeptide of the present invention (a synthetic polypeptide or a recombinant polypeptide which is a gene expression product) is subjected to an aqueous solution (for example, buffer, water) suitable for screening. Or a medium) to prepare a sample of the polypeptide of the present invention. The buffer may be a buffer that does not inhibit the contact between the polypeptide of the present invention and a nerve cell / tissue (for example, a pH of about 4 to 10, preferably a pH of about 6 to 10).
8) Phosphate buffer, Tris-HCl buffer, etc.) can be used. The contact time is usually 1 hour to 7 days, preferably about 8 hours to 3 days. Contact temperature is usually about 3
0-40 ° C. Usually, it is carried out under a 5% carbon dioxide atmosphere condition.

In the practice of the method for screening a compound that promotes or suppresses neuronal cytotoxicity, for example, a compound determined to be a compound that suppresses neuronal cytotoxicity may be a neurological disease associated with neuronal cytotoxicity. For example, as a prophylactic or therapeutic agent for Alzheimer's disease. Examples of the prophylactic or therapeutic agent for Alzheimer's disease include, for example, an antibody or antibody fragment that binds to the synthetic polypeptide or the recombinant polypeptide of the present invention, and more preferably 12-53 of the polypeptide sequence of the present invention. An antibody or an antibody fragment that recognizes the amino acid sequence at position N can be exemplified. Further, in the method of screening for a compound that promotes or suppresses neuronal cytotoxic activity of the present invention, a synthetic compound that is determined to be a compound that suppresses neuronal cytotoxic activity and is used as an agent for preventing or treating Alzheimer's disease As shown in Examples below, lithium chloride (Litium chloride), Congo Red (Congo Red), B-27 supplement (B
-27 Supplement), antioxidants and the like.

The effects of introducing a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 into nerve cells include nerve cell-damaging activity, resulting in neurofibrillary tangles, an increase in the number of dead cells of nerve cells, Nerve cell injury is specifically observed due to an increase in activity of an enzyme such as intracellular LDH or a change in morphology such as apoptosis of nerve cells.

The above-mentioned nerve cell-damaging activity is about 20% or more, preferably about 3%, as compared with the case where no candidate compound is administered.
The compound that promotes or suppresses 0% or more, more preferably about 50% or more, and still more preferably about 70% or more, can be selected as a function promoter or a function suppressor of the neuronal cytotoxic activity by the polypeptide of the present invention.

The above assay is performed using a label directly or indirectly bound to the candidate compound, or by detecting the neuronal cytotoxic activity of the polypeptide of the present invention in an assay using competition with a labeled competitor. May be. Thus, the binding of the candidate compound can be easily tested. Furthermore, whether a candidate compound promotes or suppresses the neuronal cytotoxic activity of the polypeptide of the present invention may be tested by these assays using a detection system suitable for cells in which the polypeptide accumulates. Inhibitors of activation are generally assayed in the presence of a known agonist and observe the effect of the presence of the candidate compound on activation by the agonist. In addition, the assay simply involves mixing the solution containing the candidate compound and the polypeptide to form a mixture, measuring the neuronal cytotoxic activity in the mixture, and comparing the polypeptide activity of the mixture to a standard. May be.

[0082] Low molecular weight compounds (agonists and antagonists) that bind to polypeptides are BIACORE200
0 (Markgren, PO, et al., Analytical Biochemistr
y, 265, 340-350 (1998)).

The polypeptide or the recombinant polypeptide of the present invention may be, for example, a nerve cell, a cell-free preparation,
It can also be used to identify agonists or antagonists of the polypeptide from a mixture of chemical libraries and natural products. These agonists or antagonists may be natural or modified substrates of the polypeptide of the invention, ligands, enzymes, receptors, etc.
Alternatively, it may be a structural or functional mimic of the polypeptide of the present invention (Coligan et al., Current Protocols
in Immunology 1 (2) Chapter 5 (1991)).

As another example of a method for screening for a compound that promotes or suppresses the neuronal cytotoxic activity of the polypeptide of the present invention, the synthetic polypeptide or the recombinant polypeptide of the present invention may be used for mouse, rat, etc. Comparison between administration to vertebrates (control test) and administration of vertebrates to the synthetic polypeptide or the recombinant polypeptide of the present invention and a candidate compound is performed, and changes in nervous effects on vertebrates, for example, Physiological activities such as nerve (cell) survival sustaining action, nerve elongation action, nerve regeneration action, and glial cell activation action are about 20% or more, preferably about 30% or more, more preferably as compared with the above-mentioned control test. A test compound that enhances or suppresses about 50% or more, more preferably 70% or more, can be selected. It can be selected as a function inhibitor peptides may be candidate compounds as medicament for treating Alzheimer's disease.

In order to carry out the above screening method,
A synthetic or recombinant polypeptide of the present invention,
Alternatively, the test compound is administered to a test animal by intravenous injection, subcutaneous injection, intramuscular injection, oral administration, or the like. In the case of oral administration, the dose of the polypeptide of the present invention is generally about 0.5 per day per mammal (assuming a body weight of 50 kg).
The amount is 1 to 100 mg, preferably about 1.0 to 50 mg, and more preferably about 1.0 to 20 mg. In the case of parenteral administration, the single dose varies depending on the administration subject, administration method, and the like. For example, in the case of an injection, usually, one mammal (with a body weight of 50 kg) is used in an amount of about 0.01 to 50 mg / day.
It is convenient to administer about 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection.

The test animals include, for example, human, monkey, chimpanzee, mouse, rat, rabbit, sheep, pig,
In addition to mammals such as cows, horses, cats and dogs, fish (for example, carp, salmon, herring, rainbow trout, goldfish, etc.) are used. The inhibitory effect of the synthetic polypeptide or the recombinant polypeptide of the present invention on memory formation in the brain can be determined by a known method, for example, a Morris water maze experiment [Morris,
RGM, J. Neurosci. Meth ,. 11, 47-60 (1984)]. For example, in the case of the above-mentioned in vivo screening method, the memory formation inhibitory effect of the group administered with the synthetic polypeptide of the present invention or the recombinant polypeptide alone is about 20% or more by administration of the test compound.
A test compound that is preferably inhibited by 50% or more, more preferably 70% or more can be selected as a function inhibitor of the peptide of the present invention and is effective as a candidate compound as a therapeutic agent for Alzheimer's disease.

A screening kit according to another embodiment of the present invention contains the polypeptide of the present invention (including a recombinant polypeptide) as an essential reagent component. The following configuration 1 is an example of the screening kit of the present invention.
-3 or 1-4. Configuration 1: buffer solution for measurement Configuration 2: polypeptide preparation (polypeptide of the present invention) Configuration 3: nerve cell or nerve tissue (for example, the above-mentioned nerve cell or nerve tissue is Eagle M at 10 ⁵ cells / cm ²⁾
Cultured at 37 ° C. in a culture vessel such as a multi-well plate using an EM solution under 5% carbon dioxide gas), Configuration 4: Detection reagent, labeling agent The screening using the above screening kit is as follows. Can be implemented.

[Measurement Method] The number of cells extending the nerve axon per unit field of the well to which the test compound was added was counted, and the nerve axon per unit field of the control well to which the test compound was not added was counted. Perform a significant difference test with the number of cells present.

Further, the compounds or salts thereof obtained by using the screening method or the screening kit of the present invention may be any of the test compounds described above, for example, peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, It is a compound selected from a plant extract, an animal tissue extract and the like, and is a compound that promotes or suppresses the action of the polypeptide of the present invention. The compound which suppresses the function of the polypeptide of the present invention itself exhibits a physiological activity such as a nerve (cell) survival maintenance action, a nerve elongation action or a nerve regeneration action in the central nervous system or the like. May be additively or synergistically inhibited, or may not exhibit the biological activity itself, but may exert the effect of inhibiting the effect of the polypeptide of the present invention.

Examples of the salt of the compound include salts with physiologically acceptable bases (eg, alkali metals) and acids (eg, organic acids, inorganic acids). Particularly preferred are physiologically acceptable acid addition salts. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) and organic acids (eg, acetic acid, formic acid, propionic acid,
Fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid,
Malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid).

The compounds or salts thereof which inhibit the action of the polypeptide of the present invention can be used as safe and low toxic therapeutic and preventive agents for various neurodegenerative diseases such as Alzheimer's disease, Alzheimer's dementia, cerebral ischemia and Parkinson's disease. Useful.

The compound which suppresses nerve cell injury obtained by the above screening method is useful as a drug for various neurodegenerative diseases such as Alzheimer's disease, Alzheimer's dementia, cerebral ischemia and Parkinson's disease. The medicament is usually used as a pharmaceutical composition comprising a compound as an active ingredient and a suitable carrier for treating or preventing a patient.

The compound as an active ingredient in the pharmaceutical composition also includes a pharmaceutically acceptable salt thereof.
Such salts include those prepared by methods well known in the art,
For example, sodium, potassium, lithium, calcium,
Non-toxic alkali metal salts such as magnesium, barium and ammonium, alkaline earth metal salts, ammonium salts and the like are included. Further, the above salts also include non-toxic acid addition salts obtained by reacting the polypeptide of the present invention with a suitable organic or inorganic acid. Representative non-toxic acid addition salts include, for example, hydrochloride, hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, Borate, benzoate, lactate, phosphate,
p-toluenesulfonate (tosylate), citrate, maleate, fumarate, succinate, tartrate, sulfonate, glycolate, maleate, ascorbate, benzenesulfonate and An example is napsylate.

The above-mentioned pharmaceutical compositions include those containing the compound of the present invention as an active ingredient and a pharmaceutically effective amount thereof together with a suitable nontoxic pharmaceutical carrier or diluent.

Pharmaceutical carriers or diluents which can be used in the above-mentioned pharmaceutical composition (pharmaceutical preparation) include fillers, extenders, binders, humectants, disintegrants and the like which are usually used according to the use form of the preparation. Examples include surfactants, lubricants, and excipients, which are appropriately selected and used according to the dosage unit form of the resulting preparation.

When the pharmaceutical preparation obtained by the screening method of the present invention is a protein preparation, various components which can be used for ordinary protein preparations, such as stabilizers, bactericides, buffers, isotonic agents , A chelating agent, a pH adjuster, a surfactant and the like as appropriate.

Examples of the stabilizer include human serum albumin, ordinary L-amino acids, saccharides, cellulose derivatives, and the like. These can be used alone or in combination with a surfactant. In particular, according to this combination, there are cases where the stability of the active ingredient can be further improved.

The L-amino acid is not particularly limited and may be, for example, any of glycine, cysteine, glutamic acid and the like.

The above-mentioned sugars are not particularly limited. For example, monosaccharides such as glucose, mannose, galactose and fructose, sugar alcohols such as mannitol, inositol and xylitol, disaccharides such as sucrose, maltose and lactose, dextran and hydroxypropyl Polysaccharides such as starch, chondroitin sulfate, and hyaluronic acid and derivatives thereof can be used.

The cellulose derivative is not particularly limited, and methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and the like can be used.

The surfactant is not particularly limited, and both ionic and nonionic surfactants can be used. Examples thereof include polyoxyethylene glycol sorbitan alkyl ester-based, polyoxyethylene alkyl ether-based and
Sorbitan monoacyl esters, fatty acid glycerides and the like can be used.

The amount of the saccharide to be added is about 0.0001 mg or more, preferably about 0.01 to 0.01 mg / μg of the active ingredient.
Suitably, the range is about 10 mg. The amount of the surfactant added is about 0.00001 mg per 1 μg of the active ingredient.
It is appropriate that the amount is not less than about, preferably about 0.0001 to 0.01 mg. The amount of human serum albumin to be added is about 0.0001 mg or more, preferably about 0.001 to 0.1 mg per 1 μg of the active ingredient. The amino acid is suitably used in an amount of about 0.001 to 10 mg per μg of the active ingredient.
The amount of the cellulose derivative to be added is about 0.00001 mg or more per 1 μg of the active ingredient, preferably about 0.00001 mg.
It is appropriate that the range be about 01 to 0.1 mg.

The amount of the active ingredient contained in the pharmaceutical preparation of the present invention is appropriately selected from a wide range, but is usually about 0.00000.
It is appropriate that the content is in the range of about 1 to 70% by weight, preferably about 0.0001 to 5% by weight.

In the pharmaceutical preparation of the present invention, various additives,
For example, a buffer, an isotonic agent, a chelating agent and the like can be added. Here, as a buffer, boric acid, phosphoric acid, acetic acid, citric acid, ε-aminocaproic acid, glutamic acid and / or a salt corresponding thereto (for example, alkali salts such as sodium salt, potassium salt, calcium salt, magnesium salt and the like) Metal salts and alkaline earth metal salts). Examples of the tonicity agent include sodium chloride, potassium chloride, sugars, glycerin and the like. Examples of the chelating agent include sodium edetate and citric acid.

The pharmaceutical preparation of the present invention can be used in the form of a solution. Alternatively, the preparation can be freeze-dried to a state in which it can be stored, and then dissolved in a buffer solution containing water for use, a saline solution, or the like. It is also possible to use it after adjusting it to an appropriate concentration.

As the dosage unit form of the pharmaceutical preparation of the present invention, various forms can be selected according to the purpose of treatment. Representative examples are tablets, pills, powders, powders, granules, capsules Solid dosage forms, such as solutions, suspensions,
Includes liquid dosage forms such as emulsions, syrups and elixirs which, depending on the route of administration, may further be oral, parenteral,
They are classified into nasal preparations, vaginal preparations, suppositories, sublingual preparations, ointments and the like, and can be prepared, molded or prepared according to the usual methods.

For example, in the case of molding into a tablet form, the above-mentioned preparation carrier such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, potassium phosphate, etc. Agent, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose,
Methylcellulose, binders such as polyvinylpyrrolidone, carboxymethylcellulose sodium, carboxymethylcellulose calcium, low-substituted hydroxypropylcellulose, dried starch, sodium alginate, agar powder, laminaran powder, sodium hydrogen carbonate,
Disintegrators such as calcium carbonate, surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate and stearic acid monoglyceride, disintegrators such as sucrose, stearin, cocoa butter, and hydrogenated oil; quaternary ammonium bases; Absorption promoters such as sodium lauryl sulfate, humectants such as glycerin and starch, adsorbents such as starch, lactose, kaolin, bentonite, and colloidal silicic acid; Sourcing agents and the like can be used.

Further, the tablets may be tablets coated with a usual coating, if necessary, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, and double tablets or multilayer tablets. Can also.

In the case of molding into the form of pills, pharmaceutical carriers such as glucose, lactose, starch, cocoa butter,
Excipients such as hardened vegetable oil, kaolin, and talc, gum arabic powder, tragacanth powder, binders such as gelatin and ethanol, and disintegrants such as laminaran and agar can be used.

Capsules are prepared by mixing the active ingredient of the present invention with the various pharmaceutical carriers exemplified above and filling the mixture into hard gelatin capsules, soft capsules, and the like, according to a conventional method.

Liquid dosage forms for oral administration include pharmaceutically acceptable solutions containing conventional inert diluents such as water,
It includes emulsions, suspensions, syrups, elixirs and the like, and may further contain auxiliaries such as wetting agents, emulsions and suspensions, which are prepared according to a conventional method.

In preparing liquid dosage forms for parenteral administration, such as sterile aqueous to non-aqueous solutions, emulsions and suspensions, diluents such as water, ethyl alcohol, propylene glycol, polyethylene glycol and ethoxylated Vegetable oils such as isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters and olive oil can be used, and injectable organic esters such as ethyl oleate can be blended. These may further contain ordinary solubilizers, buffers, wetting agents, emulsifiers, suspending agents, preservatives, dispersants and the like. Sterilization is, for example, a filtration operation through a bacteria retaining filter,
It can be carried out by blending a bactericide, irradiation treatment and heat treatment. They can also be prepared in the form of sterile solid compositions which can be dissolved in sterile water or a suitable sterilizable medium immediately before use.

For shaping in the form of a suppository or a preparation for vaginal administration, for example, polyethylene glycol, cocoa butter, higher alcohol, esters of higher alcohol, gelatin, semisynthetic glyceride and the like can be used as the preparation carrier.

When shaping into the form of an ointment such as a paste, cream or gel, diluents such as white petrolatum, paraffin, glycerin, cellulose derivatives, propylene glycol, polyethylene glycol,
Vegetable oils such as silicon, bentonite and olive oil can be used.

A composition for nasal or sublingual administration can be prepared by a conventional method using well-known standard excipients.

The drug of the present invention may contain a coloring agent, a preservative, a fragrance, a flavoring agent, a sweetening agent, and other pharmaceuticals, if necessary.

The administration method of the above pharmaceutical preparation is not particularly limited, and is determined according to various preparation forms, age, sex and other conditions of the patient, degree of disease and the like. For example, tablets, pills,
Solutions, suspensions, emulsions, granules and capsules are administered orally, injections are administered alone or mixed with normal replenishers such as glucose and amino acids, and administered intravenously. Intradermal, subcutaneous or intraperitoneal administration, suppositories are administered rectally, vaginal agents are administered intravaginally, nasal agents are administered intranasally, sublingual agents are administered intraorally, ointments are transdermal Is administered locally.

The amount of the active ingredient to be contained in the above pharmaceutical preparation and the dose thereof are not particularly limited, and may be selected according to the desired therapeutic effect, administration method, treatment period, patient age, sex and other conditions. It is appropriately selected from a wide range. In general,
The dose is usually about 0.0 / kg of body weight per day.
About 1 μg to 10 mg, preferably about 0.1 μg to 1 m
g, and the preparation can be administered once or several times a day.

Further, according to the present invention, when the candidate compound is a polypeptide, a more active or stable form of the polypeptide derivative or, for example, an agent that suppresses the function of the polypeptide of the present invention in vivo To develop, it is possible to make biologically active polypeptides of interest or structural analogs thereof, such as peptide agonists, peptide antagonists, peptide inhibitors, etc., with which they interact. The structural analog can be determined by, for example, X-ray crystallography, computer modeling, or a combination thereof using the three-dimensional structure of a complex of the peptide polypeptide and another protein. Information on the structure of the structural analog can also be obtained by peptide modeling based on the structure of the homologous protein.

As a method for obtaining an active or stable form of a peptide derivative as described above, for example, alanine
It is possible to analyze by scanning. The method involves substituting Ala for an amino acid residue and measuring each of the amino acid residues of the peptide in a manner that measures its effect on the activity of the peptide, thus determining regions important for the activity and stability of the peptide. Is the way. By this method, more active or stable peptide derivatives can be designed.

Thus, a drug having an action as an inhibitor, agonist, antagonist or the like of the neuronal cytotoxic activity of the polypeptide of the present invention can be designed and developed.

Such a drug is examined by using primary cultures of hippocampal neurons and examining the effect on the survival thereof (Japan. J. Pharmacol. 53, 221-227 (1990)). Transgenic mice of mutant presenilin 1 gene (Na
ture, 373, 523-527 (1995): Nature Med., 5, 560-564.
(1999)) can be evaluated by examining the effects on neurological lesions in Alzheimer's disease model animals.

The compound thus obtained can be used not only for Alzheimer's disease but also as a therapeutic drug for cerebral infarction and other neurodegenerative diseases.

According to the present invention, there is provided a transgenic mouse containing a nucleic acid sequence encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1;
By creating a mouse (mutant mouse), which part of the amino acid sequence affects the various neuronal cytotoxic activities as described above in vivo, that is, a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 Which amino acid of the expression product of the encoding nucleic acid molecule has which function in vivo can be confirmed.

This method is a technique for intentionally modifying the genetic information of an organism using a homologous recombinant gene, and can be exemplified by a method using mouse embryonic stem cells (ES cells) (Capeccchi). , MR, Science, 244, 1288-1292 (198
9)).

Incidentally, the method for producing the above mutant mouse is already a common technique for those skilled in the art,
(Tetsuo Noda, Ed., Experimental Medicine, extra edition, 14 (20) (1996), Youtosha), and a mutant mouse can be easily produced by adapting the peptide. Therefore, by applying the above technology, it is possible to design and develop a drug having an action as an inhibitor, an agonist, an antagonist or the like of a neuronal cytotoxic activity.
Further, as another invention of the present invention, the polypeptide or the recombinant polypeptide of the present invention is specifically expressed in a nerve cell by contacting the nerve cell with the nerve cell, for example, by culturing it in an appropriate medium. It is also possible to provide a method for detecting a gene or a gene exhibiting a variation in gene expression in the nerve cell. Further, according to the present invention, the expression of the gene is fluctuated by the method, and a novel,
Alternatively, a known gene or gene expression product can be provided. The detection method is, for example, a fluorescent differential display method [Liang, P., et al., Science,
257, 967-971 (1992)] and the subtraction method
No. 9-511149] or D using DNA microarray
NA chip method [Nature Genetics Suppliment, 21,3-60
(1999)], the addition of an expression product of the polypeptide or nucleic acid molecule of the present invention makes it possible to detect a change in the gene or gene expression product in cultured neurons.

[0127] These methods for detecting gene expression include, for example, those using the fluorescent differential display method.
First, after culturing the polypeptide or the recombinant polypeptide of the present invention and a nerve cell in an appropriate medium, mRNA extracted from the nerve cell, and brain, lung, liver,
CDNA is prepared from poly-A RNA or total RNA isolated from each of a plurality of human tissues such as stomach, kidney, and the like. That is, the above mRNA was treated with diethylpyrocarbonate-treated water in 3'-anquard oligo d
Mix with any primer consisting of T primer G (15MA (M is a mixture of G, A, and C)
After heating for 1 minute, DTT, dNTPs, ribonuclease
Add inhibitor and reverse transcriptase to obtain cDNA. The cDNA obtained above is used, for example, in [α- ³³ P] AT
P in the presence of P-labelled 3 'uncard primer
Perform CR amplification. After separating the PCR reaction product by electrophoresis, it is amplified again, and compared with other tissues, it is possible to confirm a PCR product having a fluctuation in expression in nerve cells. Sequencing of the cloning product with variable expression in the neurons obtained above was performed using the ABI377 automatic sequencer.
(Applied Biosystems).

When a subtraction method is used as another method of detecting gene expression, first, the polypeptide of the present invention or the recombinant polypeptide and a nerve cell are cultured in an appropriate medium, and then the nerve cell is cultured. MRNA extracted and poly-A RNA or total R isolated from multiple human tissues such as brain, lung, liver, stomach, kidney, etc.
From the NA, cDNA is prepared using reverse transcriptase. One of the two types of cDNAs including those derived from nerve cells is used as a tester cDNA and the other is used as a driver cDNA. Both of them are cut short using an arbitrary restriction enzyme such as restriction enzyme RsaI. The tester side is divided into two, and two types of separate adapters are ligated (here, no adapter is attached to the driver side cDNA). The two testers obtained are heat denatured and hybridized with an excess amount of driver. At this time, only on the tester side (expressed differentially) c
The DNA remains efficiently single-stranded. Further, the respective reaction products after hybridization are mixed, and the cDNAs remaining as single strands are hybridized with each other, and then only cDNAs having different adapter sequences at the ends are exponentially amplified by suppression PCR.
Thus, genes having differential expression are concentrated, and the addition of a desired expression product of the polypeptide or nucleic acid molecule of the present invention enables the detection of a gene whose expression is fluctuated specifically in nerve cells. The above method is performed, for example, by PCR-Select ^™ cDNA Subtracti.
It can be easily carried out using on Kit (Clontech).

When the DNA microarray method or the DNA chip method is used as another method for detecting gene expression, for example, in the case of the DNA microarray method, the polypeptide of the present invention and nerve cells are first cultured in an appropriate medium. Thereafter, mRNA extracted from the nerve cells and poly-A RNA or total RNA isolated from another human tissue such as brain, lung, liver, stomach, kidney and the like are obtained. Next, the single-stranded oligo DNA prepared in advance is directly attached to a slide glass, and a change in the expression of the gene is detected using a dual fluorescent labeling method. Two different mRNAs obtained above
Each sample was labeled with a different fluorescence and competitive hybridization was performed on the same microarray.
By measuring and comparing both fluorescences, it is possible to detect a difference in desired gene expression. This method can be easily carried out using a DNA microarray manufactured by Incyte / Shinteny.

When the DNA chip method is used, for example, the expression product of the polypeptide or nucleic acid molecule of the present invention and a nerve cell are first cultured in an appropriate medium, and then the mRNA extracted from the nerve cell is subjected to reverse transcription. CDN by enzyme
A is synthesized. 15-25m fixed on carrier
1 million of the oligonucleotide probes were converted to VIT (in vitro transc
specific oligomer on the chip, which is labeled at the time of fragmentation into cRNA by ription and the labeled R
Hybridization with NA occurs. On the other hand, a mismatched probe obtained by converting a specific oligomer into one or two bases can be synthesized on a chip as a control, hybridized simultaneously, and the amount of mishybridization can be automatically analyzed by a computer to determine the expression level. it can. This method can be easily carried out using a DNA chip manufactured by Affymetrix.

[0131] Thus, each of the above gene expression detection methods
By culturing and contacting the expression product of the polypeptide or nucleic acid molecule of the present invention with a nerve cell in an appropriate medium, and obtaining a gene or gene product whose gene expression is fluctuated in the nerve cell can be obtained by conventional genetic engineering. Can be obtained using technical techniques.

As described above, by culturing the above-mentioned polypeptide of the present invention and a neuron in a suitable medium and bringing them into contact with each other, a gene that fluctuates in the neuron is analyzed, and a gene involved in neuronal toxicity is analyzed. It is possible to search. In addition, by using a recombinant protein expressing the identified gene in Escherichia coli, animal cells, etc.,
It is possible to search for a substance that binds to the same protein and inhibits or promotes biological activity. By expressing the gene identified by the above method in animal cells or the like, it is possible to search for a substance that inhibits neurodegeneration mediated by the expressed protein. In addition, it is possible to analyze neurodegeneration and behavioral pharmacological abnormalities by preparing a genetically modified animal incorporating the gene identified by the above method. Further, it is possible to search for a substance that inhibits neurodegeneration and behavioral pharmacological abnormalities using the genetically modified animal.

For example, in a method for measuring the neuronal cytotoxic activity of the gene or the gene expression product and detecting an agonist / antagonist for the activity of the gene or the gene expression product, the method includes the steps of: Contacting the cell with a test sample, (b) detecting cytotoxicity of the nerve cell, and (c) selecting a compound that promotes or suppresses cytotoxicity of the nerve cell. A method is also provided. This method can be performed in the same manner as the above method.

[0134]

According to the present invention, a novel method for measuring a cytotoxic activity due to accumulation of a substance incorporated in a nerve cell and a method for screening a compound which affects the cytotoxic activity on a nerve cell using the assay method The use of the measurement method or the screening method makes it possible to develop a therapeutic agent based on the analysis of the neurotoxic mechanism caused by intracellular amyloid β, and particularly to find a candidate compound as a therapeutic agent for Alzheimer's disease. The development of a therapeutic agent is also useful in that the progress of Alzheimer's disease can be prevented at an early stage.

[0135]

EXAMPLES Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to this range.

Example 1 Solid-Phase Synthesis of Polypeptides The present inventors prepared a fully automatic peptide synthesizer (ACT357:
According to the company's program, using Fmoc / NMP, HOBt method [Fmoc: 9-fluorenylmethoxycarbonyl, NMP: N-methylpyrrolidone, HOBt: 1-hydroxybenzotriazole] Solid phase synthesis of the peptide was performed as follows.

That is, first, a C-terminal free (OH) peptide was produced. According to the amino acid sequence shown in SEQ ID NO: 1, the Fmoc-amino acid-Alko resin 0.25 mmol corresponding to the C-terminal amino acid was added with 2
The extension reaction was performed according to the synthesis program according to the Fmoc-amino acid corresponding to each amino acid after the first amino acid.

Each peptide of the C-terminal amide is represented by Fm
A 0.25 mmol of oc-NH-SAL resin is subjected to a condensation reaction with an Fmoc-amino acid corresponding to the C-terminal amino acid according to a synthesis program, and then a Fmoc-amino acid corresponding to each of the second and subsequent amino acids from the C-terminal is sequentially subjected to a condensation reaction to form a chain. Extension was performed.

After completion of each reaction, according to the program,
A deprotection reaction of the N-terminal Fmoc group was performed. Each peptide resin obtained in this way is mini-column made of polypropylene
(Manufactured by Assist Co., Ltd.), washed with methanol, dried in vacuum, and subjected to the following operations to cut out the peptide from the resin and to perform a side chain deprotection reaction. That is, Reagent K (82.5% TFA, 5% phenol,
2 ml of 5% H ₂ O, 5% thioanisole (2.5% ethanedithiol) was added, and the mixture was reacted in a mini column for 60 minutes.

Next, the reaction solution was cooled with 8 ml of cold diethyl ether.
1 to allow the reaction to take place, while simultaneously precipitating the peptide. In addition, the mini-column was replaced with trifluoroacetic acid (TFA).
After washing with 2 ml, 5 ml of cold diethyl ether was added, and the mixture was centrifuged. The precipitate was washed three times with 10 ml of diethyl ether, the peptide was solubilized with about 5 ml of 50% acetonitrile, and freeze-dried. Further solubilization and freeze drying
This was repeated twice to obtain a desired crude freeze-dried product.

The crude freeze-dried product obtained above was fractionated by reverse-phase high performance liquid chromatography (HPLC) using an octadecyl column (diameter: 20 × 250 mm, manufactured by YMC) to isolate a desired polypeptide. The resin and the amino acid derivative used in the above were all manufactured by Watanabe Chemical Industry Co., Ltd.

The identity of the polypeptide thus isolated was determined by amino acid sequence analysis using a gas phase protein sequencer (model 477A) with a PTH analyzer (model 12).
0A, both manufactured by Applied Biosystems). Further, the polypeptide was identified by mass spectrometry using Voyager-DE Pro (manufactured by Perkin Elmer Biosystems) by mass spectrometry. Thus, a polypeptide (TAT-Aβ42) consisting of 53 amino acids having the desired amino acid sequence represented by SEQ ID NO: 1 was chemically synthesized.

A peptide (TAT) consisting of 11 amino acids having the amino acid sequence shown in SEQ ID NO: 3 was synthesized in the same manner as described above.

Example 2: Nerve cell injury activity by synthetic polypeptide (1) Isolation and culture of hippocampal neurons [0144] The whole brain was aseptically removed from an 18-day-old fetus of an SD rat, the hippocampus was cut out, and then a female was used. After shredding, the cells are incubated at 37 ° C. for 20 minutes in a phosphate buffered saline containing 0.25% trypsin and 0.002% DNase I for enzyme treatment. After stopping the enzyme reaction by adding fetal calf serum, the operation of sucking up and discharging the cell solution with a pipette equipped with a plastic tip was repeated three times to disperse the cells. The cell solution was filtered through a filter in which two sheets of lens paper were stacked to remove undigested tissue fragments, and then 1000 rpm
For 5 minutes. The cells were then washed several times with Eagle's minimal essential medium (EMEM, Gibco BRL) and washed 10 times.
Cells were seeded at 1 × 10 ⁵ / well in a 48-well plate (manufactured by Iwakigrass) coated with poly-L-lysine (Sigma) containing EMEM medium containing 10% fetal bovine serum, and the cells were seeded at 37 ° C. and 5% CO _2. Cultured overnight under the conditions. 2% B27 supplemented with Neurobasal
Replace with medium (manufactured by Gibco BRL) at 37 ° C, 5% CO
Culture was performed under _two conditions. The medium was replaced after 3 to 4 days, and the cultured cells after 7 to 8 days of culture were subjected to the experiment. Finally, replace the Neurobasal medium with 1 × 1
0 ⁵ was plated so that the cells / cm ^2.

(2) Treatment with the Polypeptide of the Present Invention After 7 to 8 days of culture, the cells were rinsed with a neurobasal medium, and then the neurobasal medium containing the polypeptide of the present invention (TAT-Aβ42) obtained in Example 1 above. To 1, 5,
10 μM and 20 μM were added to each well (the present invention group).

For comparison, the peptide (TAT) similarly obtained in Example 1 and the peptide (Aβ42) purchased from American Peptide (Aβ protein 1-
42 parts) in a solvent (medium) in the same manner,
Was added in the same manner as above, together with 1, 5, 10 μM and 20 μM, a group consisting of only a solvent. still,
Each peptide was dissolved in distilled water at a concentration of 200 μM, diluted with Neurobasal medium, and added to the cells.

After adding the peptide, the cells were incubated at 37 ° C., 5% C
The cells were cultured under O ₂ conditions for 24 hours, and the cytotoxic activity due to the addition of the peptide was examined.

The N number in each addition amount of the polypeptide including the polypeptide of the present invention was 3 (triplet).

(3) Evaluation of Cytotoxic Activity of Polypeptide against Nerve Cells After culturing the cells (culture solution) of each group prepared in the above (2) for 24 hours, green calcein AM was used for a fluorescent dye uptake test of the cells. (CalceinAM: Dojinsha) and red propidium iodide (Propidium Iodide: Sigma) were added in an amount of 1, 20 μM each, and live cells,
The number of dead cells was measured. That is, the number of cells incorporating calcein AM or propidium iodide was measured under a fluorescence microscope, and the ratio of the number of live cells to the number of dead cells was measured. The number of cells in each of the three fields of view was counted for each well, and the ratio of the number of cells was calculated. Each value represents the mean ± standard deviation.

The results are shown in FIGS. The vertical axis in FIG. 1 shows the number of viable cells (right figure) and the number of dead cells (left figure) associated with the above treatment, and is a percentage (%) of the total number of cells measured.
Is represented by As shown in FIG. 1, when the polypeptide of the present invention (TAT-Aβ42) was allowed to act on nerve cells,
The number of surviving cells of the polypeptide (Aβ42) and peptide (TAT) was remarkably reduced, indicating that the cytotoxic activity was extremely strong. Similarly, the number of dead cells increased remarkably in the group administered with the polypeptide of the present invention, and almost 100% of the cells in the groups administered with 10 and 20 μM of the polypeptide of the present invention reached dead cells.

In FIG. 2, the vertical axis represents 10 μm of each polypeptide.
The percentage (%) of dead cells accompanying the treatment with the added amount of M is shown. still,
The number of cells before addition was set to 100%.

As shown in FIG. 2, when the polypeptide of the present invention (TAT-Aβ42) was allowed to act on nerve cells, the number of viable cells of the polypeptide (Aβ42) and the peptide (TAT) was smaller than that of the present invention. Polypeptide (TAT-Aβ42) 10 μM
In the case of the addition, it can be seen that the number of dead cells increases remarkably, and the cytotoxic activity is extremely strong. Further, when the polypeptide (Aβ42) and the peptide (TAT) are added simultaneously, the action of the polypeptide (Aβ42) is not enhanced, and the polypeptide of the present invention, which is a fusion peptide, may have strong cytotoxic activity. Understand.

FIG. 3 shows the polypeptide of the present invention (TAT-Aβ42
) 10 μM and 20 μM, polypeptide (Aβ42) 1
The fluorescence microscope image of the nerve cell in a 0 micromol and 20 micromol, peptide (TAT) 20 micromol, and a solvent (Solvent) administration group is shown. In the figure, cells stained green indicate viable cells, and cells having a smooth outline and many neurites are observed. On the other hand, the polypeptide of the present invention (TAT-Aβ42
) In the 10 μM and 20 μM administration groups, more red cells indicating dead cells were observed compared to the other groups,
In particular, in the 20 μM administration group, most of the cells stained red, which indicates that the neuronal cytotoxicity of the polypeptide of the present invention was significantly higher than that of the other polypeptide administration groups.

As a result of examining the cytotoxicity of the polypeptide of the present invention to primary cultured cells of rat fetal brain hippocampus, it was found that the polypeptide exhibited extremely high cytotoxicity as compared with the polypeptide (Aβ42). No neurotoxicity was observed with the peptide (TAT) alone. These results indicate that by accumulating the polypeptide of the present invention (TAT-Aβ42) in cells through the intervention of the peptide (TAT), a higher degree of nerve cell injury occurs, which was taken up into cells. The polypeptide (Aβ42) is the polypeptide (Aβ4
2) It is also important for neuronal degeneration caused by.

Thus, there has been provided a method for measuring a change in the cytotoxic activity on a nerve cell by introducing the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 into the nerve cell of the present invention.

Therefore, the polypeptide of the present invention comprising at least the amino acid sequence represented by SEQ ID NO: 1 (TAT-A
The analysis of the mechanism of neuronal toxicity by intracellular Aβ1-42 using β42) and the inhibition of neuronal toxicity are considered to be useful for the development of a therapeutic drug for Alzheimer's disease based on amyloid hypothesis. The accumulation of Aβ 1-42 in nerve cells is said to occur at an early stage of the disease state prior to senile plaque accumulation and neurofibrillary tangles. Therefore, treatment based on analysis of the neurotoxic mechanism by intracellular Aβ 1-42 is considered. The development of a drug is also useful in preventing the progress of a disease state early.

Example 3 Method for Screening Candidate Compounds Further, by utilizing the results of Example 2 above, nerve (cell) injury caused by the polypeptide having the amino acid sequence of SEQ ID NO: 1 of the present invention is promoted or suppressed. A method for screening compounds was performed.

That is, a known compound which has already been shown to have an effect of suppressing nerve cell injury by the Aβ 1-42 polypeptide since it has already been shown to suppress nerve cell damage in Example 2 described above, the polypeptide of the present invention (TAT-Aβ42 It was thought that if the neuronal cytotoxicity of the present invention could be suppressed, it would be possible to provide a method for screening a compound that promotes or suppresses neuronal cytotoxicity using the neuronal cytotoxicity activating action of the polypeptide of the present invention.

The B27 supplement (B27) was tested for the release of lactate dehydrogenase (LDH) and M
Aβ2 was determined by TT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromite) assay.
5-35 is known to be effective in preventing neuronal death (Huang, HM, et al., Life Sciences, 66 (19) 1
879-1892 (2000)). B27 supplement is vitamin E,
Contains antioxidants such as vitamin C, catalase, glutathione, superoxide dismutase (SOD). Congo Red, a type of dye, has also been reported to suppress Aβ-induced toxicity and amyloid peptide aggregation in PC12 neurons (Lorenzo, A., and Yankn).
er, BA, Proc. Natl. Acad. Sci. USA, 91, 12243-122.
47 (1994)).

Culture of primary cultured rat fetal brain hippocampus cells
After 8 days, cells were rinsed in Neurobasal medium. A
Lithium chloride (LiCl) 10 mM at a concentration reported to inhibit neuronal damage by β1-42 polypeptide,
Congo Red, CHROMA-GESELLSHAFT, Schm
id GmbH Co.) (CR) 5 μM or B27 (B-27
Supplement, manufactured by GIBCO BRL (2%) and 200 μl of neurobasal medium containing 10 μM of the polypeptide of the present invention (TAT-Aβ42) were added to each well. After the addition of the polypeptide of the present invention, the cells were cultured for 24 hours at 37 ° C. and 5% CO ₂ , and the number of live cells and the number of dead cells were counted. The analysis shows the results obtained by one-way analysis of variance and analysis by the Dunnett's test using the group to which the polypeptide of the present invention was added as a control. FIG. 4 shows the results. The vertical axis indicates the ratio of the number of dead cells.

As shown in FIG. 4, the ratio of the number of dead cells in the primary cultured hippocampal neurons due to the addition of the polypeptide of the present invention was as follows: lithium chloride 10 mM, Congo red 5 μM, B27
It was significantly inhibited by the addition of 2% (*: P <0.05, **: P <0.
01).

The above results show that the polypeptide of the present invention can be used in a method for screening for a compound that promotes or suppresses nerve cell injury by utilizing the effect of activating nerve cell injury.

FIG. 5 shows the polypeptide of the present invention (TAT-Aβ42
) 20 μM, lithium chloride 10 mM, polypeptide of the present invention (TAT-Aβ42) 20 μM + lithium chloride 10 mM,
4 shows fluorescence microscopic images of nerve cells in a group administered with a solvent and a solvent (Solvent). In the figure, cells stained green indicate viable cells, and cells having a smooth outline and numerous neurites were observed. On the other hand, the polypeptide of the present invention (TAT-A
(β42) In the 20 μM administration group, red cells indicating dead cells were observed in most of the nerve cells. However, in the group added in combination with lithium chloride, which is known to have a nerve cell injury inhibitory activity, the polypeptide (TAT-Aβ42)
Compared with the M-administered group, the number of dead red cells decreased, and the number of remaining viable green cells was observed more. This indicates that the neuronal cytotoxicity by the addition of the polypeptide of the present invention was significantly inhibited by the co-addition with lithium chloride.

Example 4 Production of Antibody (1) Preparation of Polyclonal Antibody: 800 μg of the purified polypeptide (TAT-Aβ42) having the amino acid sequence represented by SEQ ID NO: 1 obtained in Example 1 above was dissolved in PBS. Then, an equal volume of Freund's complete adjuvant solution is added thereto to prepare a suspension. This suspension was immunized with 8 rabbits (New Zealand white rabbits (body weight: 2.5-3.0 kg, purchased from Charles River)) with the polypeptide of the present invention (TAT-Aβ42) together with complete Freund's adjuvant. Subcutaneously or intradermally at a dose of 100 μg / rabbit, and 2 weeks after administration to these rabbits.
A second immunization is performed. At this time, an incomplete adjuvant is used instead of Freund's complete adjuvant. Booster immunizations are performed every 10 days to 2 weeks until the antibody titer rises. If a stable increase in antibody titer is observed, perform large blood sampling. After blood collection, this was subjected to centrifugation (3000 rpm, 20 minutes),
Obtain antiserum. The antiserum is further inactivated by treatment at 56 ° C. for 30 minutes.

The antiserum obtained above was prepared according to the method of Ishikawa et al.
In accordance with Immunoassay, 4, 209 (1983)), IgG was collected by performing ammonium sulfate fractionation and diethylaminoethyl-cellulose column fractionation, and the purified IgG of the polypeptide of the present invention (TAT-Aβ42) obtained in Example 1 above. ) To obtain the desired rabbit anti-polypeptide (TAT-Aβ42) polyclonal antibody.

(2) Preparation of monoclonal antibody: 5 of purified polypeptide (TAT-Aβ42) obtained in Example 1 above
Using 0 μg as an immunizing antigen, BALB / C mice (male, 7 weeks old, weight 20-25 g, purchased from SLC)
It was administered intraperitoneally with an equal volume of complete Freund's adjuvant. Every 2 to 3 weeks, the same amount was added three times with an incomplete adjuvant, and immunization was performed. Then, 1 to 2 weeks later, 50 μg of the purified polypeptide (TAT-Aβ42) was used as a final immunization.
Is administered intravenously. Three to four days after the final immunization, the spleen was excised from the mouse, the spleen cells were taken out of the excised spleen, crushed in 10 ml of RPMI-1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.), and then centrifuged (1500 rpm, 5 rpm).
), And the red blood cells present in the cells are treated with 0.83% ammonium chloride buffer for 1-2 minutes to thaw and remove. The cells obtained above are collected as sensitized lymphocyte cells, and washed three times with RPMI-1640 medium heated at 37 ° C.

Next, mouse myeloma cells [P3U1, Curr
ent Topics in Microbiology and Immunology, 73,3 (1
981)] with 15% FCS (fetal calf serum)
The cells are subcultured in a medium in which 8-azaguanine (100 μM) is added to I-1640 medium, and this is used as a myeloma cell, and washed in the same manner as the lymphocyte cell.

The spleen cells and myeloma cells were mixed in a 50 ml tube so that the cell ratio was 10: 1, and the obtained cell mixture was centrifuged at 500 × g for 5 minutes. With a Pasteur pipette and loosen the pellet well. These operations are performed in a water tank kept at 37 ° C. Next, 45% polyethylene glycol (PEG-
4000, Bering Mannheim / Yamanouchi) 2 ml was slowly added dropwise while stirring for 30 seconds, then left for 30 seconds, and then RPMI-1640 containing no FCS.
After slowly adding 5 ml of the medium over about 2 minutes and allowing to stand for 1 minute, further adding 5 ml of the same solution and allowing to stand for 3 minutes, centrifuging (1500 rpm, 5 minutes), removing the supernatant with a Pasteur pipette, The resulting pellets are mixed with 50 ml of 10% F
Cell suspension 1 × 10 in RPMI-1640 medium containing CS
Suspend to ⁵ cells / ml.

Then, the above suspension was added to a 24-well plate.
(Coaster Co., Ltd.) Dispense 1 ml / well into 4 plates, 3
7 ° C, 5% CO_TwoIn a 100% humidity incubator
And 24 hours later, 10% FC at 1 ml / well
S-added hypoxanthine 1 × 10 ^-FourM, aminopterin 4
× 10^-7M and thymidine 1.6 × 10^-FiveRPM containing M
I-1640 medium (hereinafter referred to as "HAT medium")
Add to each well. Thereafter, the supernatant was cultured on the second and third days.
Aspirate half of the ground and add the same amount of fresh HAT medium
Change the liquid. Then, change the liquid every 2-3 days.
After that, the supernatant was aspirated in the same manner on day 6, and 1 × 10^-FourM Hippo
Xanthine and 1.6 × 10^-FiveRPM containing M thymidine
I-1640 medium (hereinafter referred to as "HT medium")
I changed. Thereafter, growth is maintained in RPMI-1640 medium.
You. 7-10 days after fusion, colonies are observed with the naked eye
As the cells become larger, the cells may reach the bottom area of the 24-well plate.
The supernatant was purified from the purified polypeptide obtained in
Enzyme immunoassay using peptide (TAT-Aβ42) as an antigen (E
Tested and screened by LISA method and found positive
Immediately by limiting dilution of hybridomas
in Enzymology, 73, 3 (1981)).
You.

That is, an antigen solution diluted to 10 μg / ml in the purified immunizing antigen polypeptide (TAT-Aβ42) used for immunization was placed in a 96-well microplate for ELISA (manufactured by Nunc). 1 in each well of the plate
After dispensing 00 µl each, leaving still at 4 ° C for 2 hours to perform coating, and then removing the antigen solution, 400 µl each of 0.1% bovine serum albumin (BSA) and PBS was added.
/ Well, and allowed to stand at room temperature for 1 hour.
Remove A and PBS and add 100 μl / well of supernatant. After overnight incubation at room temperature, 0.05%
The wells were washed three times with Tween-20 and PBS, then 100 μl / well of HRP (horseradish peroxidase) -labeled anti-rabbit IgG antibody (Bio-Rad) was added and incubated for 2 hours at room temperature. After incubation, 0.0
Wash wells 5 times with 5% Tween-20 and PBS. Finally, a 100 μl / well orthophenylenediamine (OPD) substrate solution [4 mg OPD (manufactured by Sigma, production No.
P-8287), 4 μl of 30% hydrogen peroxide solution and 10 ml of citrate buffer (pH 5.0) or OPD buffer (manufactured by Sigma, No. P-4922)], and after sufficient color development, 1
The reaction is stopped by adding 50 μl of N sulfuric acid solution to each well. The color development of the substrate solution is measured by absorbance at 490 nm using an ELISA analyzer (manufactured by Titertec).

The hybridomas in the wells that are positive in the above ELISA are immediately cloned by the limiting dilution method. Specifically, Balb / c mouse spleen cells were used as feeder cells, diluted in RPMI-1640 medium supplemented with 10% FCS, and placed in a 96-well plate at 2 × 10 ⁶ cells / 100.
Prepare and dispense to make μl / well. The above EL
The cells in the wells colored by ISA were diluted with RPMI-1640 medium supplemented with 10% FCS to 3 cells / ml,
Dispense 100 μl / well into a 96-well plate.

After the clones have proliferated to a certain extent, screening is carried out again in the same manner as described above. Single clones are ascites-fed or cryopreserved, and the others are cloned again.

By repeating cloning until all the wells of one colony become positive, a hybridoma producing the monoclonal antibody of the present invention having a desired reaction specificity can be obtained.

[0174]

[Sequence List] SEQUENCE LISTING <110> OTSUKA PHARMACEUTICAL CO., LTD. <120> A METHOD OF TESTING FOR CYTOTOXICITY <130> PR-299 <140> <141> <160> 4 <170> PatentIn Ver. 2.1 <210 > 1 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: fusion polypeptide of HIV-TAT protein 47-57 and A Beta protein 1-42 <400> 1 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Asp Ala Glu Phe Arg 1 5 10 15 His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala 20 25 30 Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly 35 40 45 Gly Val Val Ile Ala 50 <210> 2 <211> 165 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Coding DNA of SEQ. No. 1 <220> <221> CDS <222> (1) .. (162) <400> 2 atg tat ggc agg aag aag cgg aga cag cga cga aga gat gca gaa ttc 48 Met Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Asp Ala Glu Phe 1 5 10 15 cga cat gac tca gga tat gaa gtt cat cat caa aaa ttg gtg ttc ttt 96 Arg His Asp Ser Gly Tyr Glu Val Hi s His Gln Lys Leu Val Phe Phe 20 25 30 gca gaa gat gtg ggt tca aac aaa ggt gca atc att gga ctc atg gtg 144 Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val 35 40 45 ggc ggt gtt gtc ata gcg tag 165 Gly Gly Val Val Ile Ala 50 55 <210> 3 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: HIV-TAT protein 47-57 <400 > 3 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 4 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: A Beta protein 1-42 <400> 4 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 15 Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30 Gly Leu Met Val Gly Gly Val Val Ile Ala 35 40

[Brief description of the drawings]

FIG. 1 shows the cytotoxic activity of each peptide containing the polypeptide of the present invention. The vertical axis in FIG. 1 shows the number of surviving cells (right panel) and the number of dead cells (left panel) associated with the above treatment, and is expressed as a percentage (%) of the total cell number measured.

FIG. 2 shows the cytotoxic activity of each peptide containing the polypeptide of the present invention. The vertical axis in FIG. 2 shows the percentage (%) of dead cells due to the treatment of each polypeptide at an addition amount of 10 μM. In addition, the number of cells before addition was set to 100%.
The results obtained by performing a one-way analysis of variance and analyzing the results by Dunnett's test using the group without administration of the polypeptide as a control and one-way analysis of variance between the groups to which each polypeptide was administered are shown.

FIG. 3 shows the polypeptide of the present invention (TAT-Aβ42).
10 μM and 20 μM, polypeptide (Aβ42) 10
The fluorescence microscope image of the nerve cell in a micromolecule and 20 micromol, peptide (TAT) 20 micromol, and a solvent (Solvent) administration group is shown. In the figure, cells stained green indicate viable cells, and red cells indicate dead cells.

FIG. 4 is a graph showing the inhibitory effect of the polypeptide of the present invention on cytotoxic activity. The analysis shows the results obtained by one-way analysis of variance and analysis by the Dunnett's test using the group to which the polypeptide of the present invention was added as a control.

FIG. 5 shows the polypeptide of the present invention (TAT-Aβ42).
The fluorescence microscope image of the nerve cell in the administration group of 20 μM, 10 mM lithium chloride, 20 μM of the polypeptide of the present invention (TAT-Aβ42) +10 mM lithium chloride, and the solvent (Solvent) is shown. In the figure, cells stained green indicate viable cells, and red cells indicate dead cells.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 25/16 A61P 25/28 4C085 25/28 C07K 14/16 4H045 C07K 14/16 14/47 14/47 16/10 16/10 16/18 16/18 19/00 19/00 C12Q 1/02 C12Q 1/02 G01N 33/15 Z G01N 33/15 33/50 Z 33/50 C12P 21/08 // C12P 21 / 08 C12N 15/00 ZNAA (72) Inventor Masato Horie 261 F-term (reference) 2G045 AA40 BB20 DA36 FB01 FB03 FB06 GC15 4B024 AA01 AA11 BA80 CA01 4B063 QQ61 QQ79 QR02 QR41 QR48 QR77 QS36 QX01 4B064 AG26 AG27 CA10 CA20 CC24 DA01 DA13 4C084 AA17 MA01 NA14 ZA012 ZA152 ZA162 ZA392 ZB212 4C085 AA14 CC32 EE01 GG01 4H045 AA10 AA11 AA30 BA16 BA19 BA20 BA41 CA05 CA21 AE50 GA25

Claims (17)

[Claims] 1. A method for measuring a nerve cell-damaging activity by introducing a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 into a nerve cell. 2. The method of claim 1, wherein the cytotoxic activity is morphologically altered and / or
Or the method for measuring cytotoxic activity according to claim 1, which is measured by the number of living or dead cells. 3. The method for measuring cytotoxic activity according to claim 1, wherein the nerve cell is a primary cultured cell of a cortical nerve of the brain. 4. A method for screening for a compound that promotes or suppresses a nerve cell-damaging activity by introducing a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 into a nerve cell. 5. A method comprising introducing a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 into a nerve cell,
A method for screening an Alzheimer's therapeutic / prophylactic agent. 6. A method for screening for a compound that promotes or suppresses a nerve cell-damaging activity by introducing a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 into a nerve cell, wherein (a) the method comprises: Contacting the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 alone or the polypeptide and a test sample together, and (b) detecting the cell injury of the nerve cell in both of the above (a). And (c) selecting a compound that promotes or suppresses the cytotoxicity of the nerve cells. 7. The method according to claim 6, wherein the cytotoxic activity is measured by morphological changes and / or the number of living or dead cells. . 8. The method for screening for a compound that promotes or suppresses neuronal cytotoxicity according to claim 6, wherein the nerve cell is a primary cultured cell of cortical nerve of the brain. 9. The compound which suppresses a nerve cell-damaging activity by introducing a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 into a nerve cell is an antibody that binds to the polypeptide or an antibody fragment thereof. The screening method according to any one of claims 1 to 8. 10. A synthetic polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1. 11. A nucleic acid molecule encoding the polypeptide of claim 10. 12. A recombinant polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1. 13. A nerve cell-damaging agent comprising a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1. 14. An antibody or an antibody fragment thereof that binds to the polypeptide of claim 10 or 12. 15. A recombinant polypeptide, wherein the polypeptide used in the method according to any one of claims 1 to 9 is expressed by a nucleic acid molecule encoding the polypeptide of claim 11. 16. A compound which suppresses neuronal cytotoxicity obtained by the method for screening a compound according to claim 4. 17. A therapeutic or prophylactic agent for Alzheimer's disease, comprising the compound of claim 16 as an active ingredient.