WO1999051752A1 - CELL LINE NOT EXPRESSING β-SECRETASE ACTIVITY - Google Patents

Abstract

A cell which produces a β-amyloid precursor protein, expresses the α-secretase activity and does not express the β-secretase activity in the absence of any stimulus but expresses the β-secretase activity under a stimulus. This cell is useful in cloning a DNA encoding a protein having the β-secretase activity.

Description

 Specification

^ Cell lines that do not express secretase activity

Field of the invention

 The present invention relates to a cell line that does not express ^ -secretase activity. In addition, the present invention relates to a method for cloning cDNA encoding β-secretase using a cell line that does not express 3-secretase activity. Background art

 Alzheimer's disease is a progressive degenerative disease of the central nervous system, a dementia characterized by pathological features of senile plaques deposited extracellularly and neurofibrillary tangles in nerve cells. In particular, senile plaques are widespread only in Alzheimer's disease and Dunn's syndrome, and have high disease specificity. It is known that the amount of senile plaques and dementia are correlated in Walzheimer's disease (Cummings, BJ and Cotman, CW Lancet (1995) 346, 1524-1528).

Amino acid 39-42 amyloid beta peptides (A) were identified as the main components of senile plaques (Genner, GG and Wong, C.W. Biochem, Biophys. Res. Commun. (1984) 120, 885-890). In addition, / S-amyloid precursor protein (APP) was strongly cloned as a precursor of A / S (Kang, J. et al Nature (1987) 325, 733-736). APP is a type I membrane protein with one transmembrane site, and alternative splicing results in APP695, APP751 and APP770 proteins consisting of 695, 751 and 770 amino acids, respectively. A / 3 is located near the C-terminus of the APP. From the N-terminus of k & to the amino acid at position 28, A is outside the transmembrane site of APP. The region from the amino acid at position 29 in / 3 to the C-terminus is within the transmembrane site of APP.

Mutations in the APP gene have been found in familial Alzheimer's disease (Goate, A. et al., Nature (1991) 349, 704-706; ullan, M. et al., Nature Genet. (1992) 1, 345-347, Hendriks, L. et al., Nature Genet. (1992) 1, 218-221). The mutation is located near the N-terminus or C-terminus of AS or within AyS. Therefore, it is thought that the generation of AS from APP and the deposition of A3 in the brain are deeply involved in the development of Alheimer's disease.

 It is known that there are at least two different pathways of APP metabolism (Estus, S. et al., Science (1992) 255, 726-728). The first is a metabolic pathway that is cleaved between Lys at position 612 and Leu at position 613 of APP695. In this metabolic pathway, the secretion form of APP containing a part of A3 is cleaved between amino acids at position 16 and 17 at the amino acid inside the A / S peptide. Generates N-terminal fragment (sAPPa). The protease activity involved in this metabolic pathway is called α-secretase activity.

 The second is cleavage between the amino acid Met at position 596 of ΑΡΡ695 and the amino acid Asp at position 597, and between the amino acid Ala at position 638 and the amino acid Thr at position 639 of APP695. Is the metabolic pathway that is performed. In this metabolic pathway, cleavage occurs at positions before and after Αβ, and 分泌 3-free 分泌 -secreted Ν-terminal fragment (sAPP ^) and A are generated, and the C-terminal fragment of A3 is converted to a membrane. Left on. The protease activity that cleaves the N-terminus of A3 is called —secretase activity, and the protease activity that cleaves the C-terminus of A3 is called y—secretase activity.

It has been reported that both sAPPα and sAPP / 3 are secreted by these two metabolic pathways in the cultured Itoda spore line (teratocarcinomal P19 eel K embryonic kidney 293 cells) (Libin , H. et al., J. Biol. Chem. (1996) 271, 30929-30934). / S—Secretase activity and secondary secretion pathway via the secretase activity

Because of the generation of A3, it is considered important to study this metabolic pathway to elucidate the pathogenesis of Alzheimer's disease and to develop therapeutic drugs.

 To date, no protease involved in the two metabolic pathways of APP has been identified. As enzymes exhibiting the activity of cleaving APP near the N-terminus, chymotrypsin-like proteases (Nelson, RB et al,. J. Neurochem. (1993) 61, 567-577) and cathepsin D ( Ladror, US et al., J. Biol. Chem. (1994) 269, 18422-18428), but the identity of 3-secretase has not yet been elucidated.

 All of the cell lines reported so far have the ability to have both α-secretase activity and 3-secretase activity <, and cell lines that have only one secretase activity and do not have S-secretase activity It was not known. Disclosure of the invention

 An object of the present invention is to provide a cell line that does not express / 3-secretase activity. The present invention also provides a method for cloning the S-secretase gene.

That is, the present invention provides (A) a cell line that produces (l) APP, (2) expresses α-secretase activity, and (3) does not express 3-secretase activity under no stimulation. I will provide a. The cell line preferably expresses detectable secretase activity under stimulus. The cell line is preferably an animal cell line. The cell line is more preferably a mammalian cell line. As a mammal, it is preferably a human. This cell line is Or it is derived from megakaryoblastic cells.

 The present invention also relates to (B) introducing a DNA library into the cell line of (1) and (A), selecting (2) expressing DNA, and selecting (3) cells expressing the 3-secretase activity. And (4) isolating DNA encoding a protein having a 3-secretase activity from the selected cells, and providing a method for cloning DNA encoding secretase. I do. The DNA library to be introduced is preferably a cDNA library. The cDNA library is preferably a cDNA library from a mammalian cell. The cDNA library is preferably a brain-derived cDNA library.

The present invention also provides (C) (1) preparing DNA from the cell of (A) without stimulation, (2) preparing DNA from the cell of (A) under stimulation, (3) Obtaining a DNA clone which is not present in the obtained DNA and which is present in the DNA obtained in (2), and ( ₄ ) isolating a DNA encoding a protein having a secretase activity. Provide a method for cloning DNA encoding S-secretase. The DNA to be prepared is preferably cDNA.

 The present invention also provides (D) a DNA encoding a protein having 3-secretase activity obtained by the cloning method described in (B) or (C) above. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 schematically shows the protease activity of α-secretase, 3-secretase and α-secretase on ΑΡΡ. An upward black triangle indicates a cut site.

FIG. 2 is a diagram showing the results of Westin analysis of sAPP in the culture supernatant of megakaryoblastic cells. The numerical value on the vertical axis indicates the molecular weight, and the arrow Represents the position of sAPP.

 FIG. 3 shows the results of Western analysis of sAPP in the culture supernatant of megakaryoblastic cells stimulated by phorbol ester. A shows the results for MK / A P 8 cells, and B shows the results for MKZNL cells

. Numerical values on the vertical axis represent molecular weight.

 FIG. 4 shows the results of enzymatic immortal assay on sAPP in the culture supernatant of SH-SY5Y cells. The vertical axis represents the absorbance at 450 nanometers, and the horizontal axis represents the dilution ratio of the culture supernatant.

 FIG. 5 shows the results of enzymatic immobilization of sAPP in the culture supernatant of megakaryoblastic cells stimulated by phorbol ester. The vertical axis represents the absorbance of 450 nanometers, and the horizontal axis represents the number of days of phorbol ester stimulation. Embodiment of the Invention

 The present invention relates to a cell strain which (A) produces (1) APP, (2) expresses -secretase activity, and (3) does not express /?-Secretase activity under no stimulation. The cell line of the present invention does not express 5-secretase activity in a non-stimulated state, but does not express a differentiation inducer such as phorbol ester, interleukin-3 (IL-3), retinoic acid and the like. When stimulated — express secretase activity. The stimulated cell line of the invention expresses detectable secretase activity. In addition, the cell line of the present invention may be expressing a secretase activity.

The cell line of the present invention is a cell line derived from an animal or the like, and is preferably an animal cell line. The animal cell line is preferably a cell line derived from a mammal. The mammal is preferably a human-derived cell line. The cell line of the present invention may be derived from any organ, tissue or organ as long as it has the properties described above, but is preferably derived from megakaryoblastic cells. Is coming.

 The megakaryoblastic cell line MK / AP8 having the properties of the cell line of the present invention was established on March 9, 1998 by It has been deposited internationally under the Budapest Treaty under the accession number FERM BP-6287 at Chome 1-3.

 The cell line of the present invention may produce APP naturally or may be produced by genetic engineering. The cell line of the present invention is preferably a cell line that has been engineered to produce APP.

 The APPC / 3-amyloid precursor protein produced by this cell line is known to give rise to APP695, APP751 and APP770 mainly consisting of 695, 751 or 770 by alternative splicing, respectively. ing . The cell line of the present invention may produce any APP as long as it contains the amino acid sequence of A / 3. As APP, for example, the amino acid sequence of APP695 consisting of 695 amino acid residues and the corresponding base sequence are shown in SEQ ID NO: 1, and only that amino acid sequence is shown in SEQ ID NO: 5. It is shown in APP695 is a transmembrane region from the amino acid Gly at position 625 to the amino acid Leu at position 648.

APP is known to have a point mutation (FAD mutation) associated with the development of some familial Alzheimer's disease (FAD). As for the FAD mutation, in the case of APP695, as the FAD double mutation, the amino acid Lys at position 595 is Asn, the amino acid Met at position 596 is Leu, and the amino acid at position 596 is Leu, and the amino acid at position 642 is FAD. Ala at position 617 has been changed to G1 as a FAD mutation in any of Phe, Gly or IIe, and A3 within the acid. However, the APP produced by the cell line of the present invention may be an APP having all or some of these mutations. In the present invention, the APP produced by the cell line of the present invention is a biological activity that produces sAPPa by α-secretase activity and produces sAPP / 5 by S-secretase activity expressed in a stimulated state. It is a protein having Therefore, the APP produced by the cell line of the present invention is an amino acid ranging from the amino acid Leu at position 18 to the amino acid Asn at position 695 in the amino acid sequence shown in SEQ ID NO: 1 or 5. Any protein can be used as long as it has a noic acid sequence and has the biological activity of APP.

. In SEQ ID NO: 1 or 5, the signal sequence from the amino acid at position 1 Met to the amino acid at position 17 Ala is a signal sequence. In addition, APP has a deletion of one or more amino acid residues in the amino acid sequence from amino acid Leu at position 18 to Asn of amino acid at position 695 in SEQ ID NO: 1. APP having an amino acid sequence modified by addition, addition and / or substitution with another amino acid.

 APP is considered to show its biological activity if it has an amino acid sequence from amino acid lie at position 591 to amino acid Leu at position 648 in SEQ ID NO: 1 or 5. . Therefore, APP used in the present invention comprises an amino acid sequence from amino acid lie at position 591 to amino acid Leu at position 648 in SEQ ID NO: 1 or 5, and One or more residues of amino acids from the amino acid sequence from amino acid Leu to amino acid Glu at position 590 and amino acid Met at position 649 to amino acid Asn at position 695 The APP may have an amino acid sequence that has been modified by deletion, addition and / or substitution of another amino acid. In the SEQ ID NO: 1 or 5, APP is an amino acid sequence from amino acid Met at position 1 to amino acid Ala at position 1, or one or more amino acid sequences relative to the amino acid sequence. An amino acid sequence modified by deletion and addition of an amino acid residue and replacement with Z or another amino acid may be added to the N-terminus.

In addition, as long as the APP has its biological activity, SEQ ID NO: 1 or Is the deletion or addition of one or more amino acid residues to the amino acid sequence from amino acid lie at position 591 to amino acid Leu at position 648 in 5, and Z or other amino acids. An APP having an amino acid sequence modified by substitution with an amino acid may be used.

 These APPs may have an amino acid sequence having the chimera structure of APP with other membrane proteins. The amino acid sequence consisting of 42 amino acid residues from the amino acid Asp at position 597 to the amino acid Ala at position 638 of APP695 shown in SEQ ID NO: 1 or 5 is represented by A / 3. Applicable.

 A protein having an amino acid sequence modified by deletion or addition of one or more amino acid residues with respect to a certain amino acid sequence and / or substitution with another amino acid can be used as a biological protein. It is already known that the active activity is maintained (Mark, DF et al., Proc. Natl. Acad. Sci. USA (1984) 81, 5662-5666, Zoller,. J. & Smith, M. Nucleic Acid s Research (1982) 10, 6487-6500, Wang, A. et al., Science 2 24, 1431-1433, Dal bad ie-McFar 1 and, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413).

 The APP produced by the cell line of the present invention depends on the species of origin, the host producing them, and the Z or purification method, depending on the amino acid sequence, molecular weight, isoelectric point, presence or absence of glycosylation, The position, sugar chain structure, phosphorylation state, and the presence or absence of no or disulfide bonds are different. However, the APP may have any structure as long as the cell line of the present invention can be produced. Humans are the preferred species from which APP is derived.

The APP produced by the cell line of the present invention may also be the above APP fused to another peptide or polypeptide. Known methods can be used for producing these fusion polypeptides. Other peptides or polypeptides to be fused with APP may be any peptides or polypeptides produced by the cell line of the present invention. You can.

 For example, peptides include FLAG (Hopp, TP et al., BioTechnology (1988) 6, 1204-1210), 6 His (histidine) residues, 6 x His, 10 X His, and Influenza agglutinin (HA), human c-myc fragment, VSV-GP fragment, pl8HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, and -tubu 1 in A known peptide such as a fragment of B-tag or a fragment of Protein C is used. Examples of polypeptides include, for example, GST (glutathion 'S' transferase), HA (influenza agglutinin), immunoglobulin constant region, 3_galactosidase, MBP (maltose). Binding protein) and the like. A commercially available product obtained by fusing a DNA encoding these can be used.

 Examples of the DNA encoding APP produced by the cell line of the present invention include a base sequence consisting of base A at position 148 to base G at position 2235 in the base sequence shown in SEQ ID NO: 1.

 The DNA encoding APP produced by the cell line of the present invention may be any DNA as long as it has the nucleotide sequence shown in SEQ ID NO: 1. Examples of such DNA include diogenic DNA, cDNA, and synthetic DNA. These may be cDNA libraries obtained from various cells, tissues or organs, or species other than humans, or DNA obtained from the genetic library.

DNA encoding APP produced by the cell line of the present invention can be isolated using genetic recombination techniques as described above. For example, a recombinant protein can be prepared by cloning the nucleotide sequence of the APP gene described herein from a cell, tissue, or organ that expresses them, and incorporating it into an appropriate vector. Can be introduced into the host and produced. Specifically, mRNA encoding the gene is isolated from cells, tissues, or organs expressing APP. mRNA can be isolated by known methods, for example, guanidine ultracentrifugation (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), AGPC method (Chomczynski, P. and Saccchi, N.C. , Anal. Biochem. (1987) 162, 156-159) and prepare total RNA, and purify mRNA from total RNA using mRNA Purification Kit (Pharmacia). Alternatively, mRNA can be directly prepared using QuickPrep mRNA Purification Kit (Pharmacia) .o

 CDNA of the gene is synthesized from the obtained mRNA using reverse transcriptase. cDNA can also be synthesized using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (manufactured by Seikagaku Corporation). In order to synthesize and amplify cDNA, a 5'-RACE method (Frohman, MA et al.) Using Marathon cDNA Amplification kit CCLONTECH) and a polymerase chain reaction (PCR) was used. Acad. Sci. USA (1988) 85, 8998-9002, Belyav sky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932) can be used.

 Prepare the desired DNA fragment from the obtained PCR product, and ligate it with vector DNA. Further, a recombinant vector is prepared from this, and introduced into Escherichia coli or the like, and a colony is selected to prepare a desired recombinant vector. The nucleotide sequence of the target DNA is confirmed by a known method, for example, the dideoxynucleotide titration method. Once the desired DNA is obtained, incorporate it into the expression vector.

The DNA encoding APP produced by the cell line of the present invention is a DNA that hybridizes to the nucleotide sequence shown in SEQ ID NO: 1 and encodes APP having the biological activity. Is also good. Call APP Examples of the conditions under which the DNA to be hybridized are DNAs that hybridize under an appropriate stringency condition. Such hybridization conditions include, for example, low stringency conditions. The low stringency conditions include, for example, cleaning conditions given by 42 ° C., 5 × SSC, 0.1% sodium dodecyl sulfate. More preferably, high stringency conditions are included. The high stringency conditions include, for example, the cleaning conditions provided by 60 ° C., 0.1 × SSC, and 0.1% sodium dodecyl sulfate. It is already known that a protein encoded by a DNA that hybridizes under appropriate conditions to a nucleotide sequence encoding a protein has the same biological activity as the protein.

 The DNA encoding APP produced by the cell line of the present invention can be obtained by modifying the above-described DNA by a commercially available kit known in the art. For example, digestion with restriction enzymes, insertion of synthetic oligonucleotides—appropriate DNA fragments, addition of linkers, initiation codons (ATG) and / or termination codons (ATT, TGA or The expression vector of APP produced by the cell line of the present invention, which includes insertion of TAG), may be any expression vector as long as it is an expression vector suitably used in the present invention. Examples of the expression vector include a mammalian expression vector, for example, pEF, pCDM8, an insect cell-derived expression vector such as pBacPAK8, a plant-derived expression vector such as ρΜΗ1, pMH2, or an animal virus Expression vectors, e.g., pHSV, pV, expression vectors derived from yeast, e.g., pNVll, expression vectors derived from Bacillus subtilis, e.g., pPL608, pKTH50, expression vectors derived from E. coli, e.g., pGEAPP, pGE EAPP.pMALp2 o

The expression vector for APP produced by the cell line of the present invention includes, for example, APP Can be produced by ligating a DNA encoding the gene downstream of the promoter in an expression vector. Examples of promoters and enhancers include mammalian Z promoters, for example, EF1-promoter / enhancer, actin promoter // enhancer, and insect virus promoter Z enhancers. For example, polynuclear (polyhedrin) promoters, plant-derived promoters, such as plant-derived promoters, such as tobacco mosaic virus promoters, and animal virus-derived promoters. Enhancers, such as the SV40 motor, Z Enhansa, human CMV promoter / enhancer, promoters from yeast — Z enhancers, such as the alcohol dehydrogenase promoter / enhancer, E. coli-derived promoter Z Enhancer, such as the Lac promoter Z Nha Mono-, Trp promoter Z Enha Nsa, the Tac promoter / Enhansa one like.

 For expression of APP produced by the cell line of the present invention, a signal sequence suitable for the host used for expression may be added and used. Examples of the signal sequence include a signal sequence of a secretory protein. Examples of the signal sequence of a secretory protein include, for example, a signal sequence of a secretory protein derived from a mammal, for example, a signal sequence of imnoglobulin. Examples of the signal sequence of a secretory protein include a signal sequence of a secretory protein derived from Escherichia coli, for example, a periplasmic secretory signal sequence such as OmpA.

The expression vector thus prepared can be introduced into a host by a known method. Methods for introduction into a host include, for example, electroporation, the calcium phosphate method, and the ribosome method. The replication origin of the expression vector is SV40, polio-millivirus. , Adeno Wireless, ゥ Shino. It is possible to use one derived from pi0-mavirus (BPV) or the like. In addition, the expression vector should be selected in order to increase the number of gene copies in the host cell system, such as the aminoglycoside phosphotransferase (APH) gene and thymidine kinase (TK). ) Gene, Escherichia coli xanthinguanine phosphoribosyltransferase (HPRT) gene, dihydrofolate reductase (DHFR) gene, and the like. In addition, sequences with higher expression efficiency can be designed in consideration of the codon usage of the host used for expression (Gran tham, R. et al., Nucleic Acids Research (1981) 9, r43- r74) As the cell line of the present invention, any cell line can be used. Eukaryotic cells are examples of such cell lines.

 When using eukaryotic cells, there are animal cells, plant cells, and fungal cells. Examples of animal cells include (1) mammalian cells, such as CHO (J. EAPPp. Med. (1995) 108, 945), COS, myeloma, BHK (baby hamster kidney), HeLa, Vero, megakaryoblast Lineage cells, (2) amphibian cells, for example, African frog frog oocytes (Valle, et al., Nature (1981) 291, 358-340), or (3) insect cells, such as sf9, sf21, Tn5 It has been known. Examples of CH0 cells include dhfr-CHO (Proc. Natl. Acad. Sci. USA (1980) 77, 4216-4220) and CHO K-1 (Proc. Natl. Acad. Sci. USA (1968) 60, 1275) can be suitably used.

As plant cells, cells derived from Nicotiana tabacum are known, and callus culture may be used. Fungal cells include yeast, for example, the genus Saccharomyces, for example, Saccharomyces * cerevisiae, filamentous fungi, for example, the genus Aspergillus, for example, Spergius niger (Aspergillus niger) is known. These cells are transformed with an expression vector containing a gene encoding APP, and cells that produce APP can be obtained by culturing the transformed cells in vitro. The culture is performed according to a known method. For example, DMEM, MEM, RPMI1640, IMDM can be used as a culture solution. At this time, a serum sample such as fetal calf serum (FCS) can be used in combination, or serum-free culture may be performed. The pH during culture is preferably about 6-8. Culture is usually performed at about 30-40 ° C for about 15-200 hours, and if necessary, the medium is replaced, aerated, and agitated.

 The metabolic pathway of APP consists of (1) a pathway that produces soluble APP terminal fragment (sAPP) upon cleavage by α-secretase and (2) APP solubility by 3-secretase and α-secretase. There is an N-terminal fragment (sAPP3) and a path that generates A.

 One secretase has the activity of cleaving between Lys at position 612 and Lys at position 613 of APP695 to generate sAPP. Normal cells have α-secretase activity.

 / 3-—Secretase has the activity of cleaving the 末端 -terminal side of A. β-secretase has the activity of cleaving between the amino acid Met at position 596 and the amino acid Asp at position 597 in ΑΡΡ695 to generate sAPP3. The cleavage site of ΑΡΡ by β-secretase is fixed. S-secretase produces A / 3 by acting on と 共 に with α-secretase. Normal cells have secretase activity. When APP is cleaved by one secretase, 3-secretase does not act on APP.

7—Secretase has the activity of cleaving the C-terminal side of It is known that the _7- secretase activity moves around the cleavage site.

Therefore, the amino acid at the C-terminus of the excised is heterogeneous . Therefore, multiple enzymes may exist as 7-secretases. As an example of a secretase, cleavage between the amino acid Thr at position 638 of APP695 and the amino acid Val at position 639 generates a polypeptide containing sAPP3 and β. Those having an activity to cause them are known. The arc secretase produces Α to Α 3 by acting together with 3-secretase. Normal cells have 7—secretase activity ο

 The protease activities of monosecretase, 3-secretase and alpha-secretase against ΑΡ are schematically shown in FIG.

 Α3 is 39-42 amino acids extracted from ΑΡΡ. 3-Cleaved by secretase Ν The terminus is fixed, but the C-terminus cut by the secretase is heterogeneous. Therefore, it is known that 数 ^ differs in the number of amino acid residues, but the present invention may have any number of amino acid residues. The amino acid sequence of A3 having 42 amino acid residues is shown in SEQ ID NO: 2.

 The present invention relates to (1) introducing a DNA library into the above-mentioned cell line, (2) expressing the introduced DNA, and (3) selecting a cell that expresses / 3-secretase activity; ) A method for cloning a DNA encoding a / 3-secretase, comprising isolating DNA encoding a protein having a / 3-secretase activity from selected cells.

 The DNA library to be introduced may be a cDNA library or a genomic DNA library obtained from various cells, tissues or organs. These DNA libraries may be commercially available DNA libraries. As a vector used for these libraries, any vector such as plasmid, bacteriophage, and YAC vector may be used.

In order to create a cDNA library, specifically, desired cells and tissues Or mRNA from the organ. The mRNA can be isolated by known methods, for example, guanidine ultracentrifugation (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), and the AGPC method (Chomczynski, P. and Sacchi, N. et al. Prepare total RNA according to Analytical Biochem. (1987) 162, 156-159), and purify total RNA mRNA using mRNA Purification Kit (Pharmacia). Alternatively, mRNA can be directly prepared by using the QuickPrep mRNA Purification Kit (Pharmacia).

 CDNA is synthesized from the obtained mRNA using reverse transcriptase. cDNA synthesis is performed using AMV Reverse Transcritase First-strand cDNA Synthesis.

Kit (manufactured by Seikagaku Corporation) can also be used. In order to synthesize and amplify cDNA, 5'-RACE method (Frohman, MA et al.) Using Marathon cDNA Amplification kit (CLO NTBCH) and polymerase chain reaction (RCR) was used. Acad. Sci. USA (1988) 85, 8998-9002; Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932) can be used.

 A cDNA fragment may be prepared from the obtained PCR product and ligated with the desired expression vector DNA.

 The cDNA library is preferably a mammalian cDNA library. Obtain cDNA libraries from mammalian cells, organs, tissues, and organs. As a mammal, it is preferably a human. The source of the cDNA library is preferably human brain.

As described above, / 3—secretase activity is the activity of cleaving sAPP3 from APP. Therefore, cells that express 3-secretase activity can be selected by detecting or measuring sAPP3 in the culture supernatant of the cells. sAPP3 can be detected or measured in a purified state or in a partially purified state. First, the purified or crudely purified sAPP3 is bound to a support. In binding sAPP3 to the support, sAPP ^ can be bound to the support in a standard manner. Supports to which sAPP3 is bound include, for example, insoluble polysaccharides, such as agarose, dextran, cellulose, synthetic resins, such as polystyrene, polyacrylamide, and silica. And the like. More specifically, commercially available beads and plates manufactured using them as raw materials are used. In the case of beads, a column or the like filled with these may be used. In the case of a plate, a multi-well plate (such as a 96-well multi-well plate) or a biosensor search can be used.

 The binding between sAPP / 3 and the support may be achieved by a commonly used method such as chemical bonding or physical adsorption. Alternatively, the antibody can be bound by binding an antibody that specifically recognizes sAPP3 to a support in advance, and binding this antibody to sAPPyS. Furthermore, they can be linked via avidin Z-biotin.

 Detection or measurement of sAPP / 3 is usually performed in a buffer. As the buffer, for example, a phosphate buffer, a Trans buffer, or the like is used. As the conditions for the incubate, incubation is carried out for 1 hour to 24 hours at a condition that is already widely used, for example, at 4 ° C. to room temperature. The post-incubation wash may be of any type as long as it does not hinder the binding between sAPPS and the support. For example, a buffer containing a surfactant is used. As the surfactant, for example, 0.05% Tween 20 is used.o

To detect or measure sAPP3, specific binding and non-specific binding can be separated by incubation and washing under appropriate conditions. Then, sAPP / S may be detected or measured. In this method, control is performed together with a group for detecting or measuring sAPP3 in the sample. May be installed. As the control group, a negative control group containing no sAPP / 3, a positive control group containing SAPP, or both groups can be used.

 In the present invention, when sAPP / 3 is detected or measured, sAPP3 may be simply detected, or SAPP may be quantitatively measured. In these cases, the results obtained with the negative control group without sAPP / 3, the results obtained with the sample, and the results obtained with the positive control group containing Z or sAPPyS were compared. The target sAPP; S can be detected.

 By obtaining these results as numerical values and comparing those numerical values, sAPP3 can be quantitatively measured. In the case of quantitative measurement, quantification can be performed based on a standard curve created from numerical values obtained from a positive control group containing a known amount of sAPP3.

 In the present method, a biosensor utilizing the surface plasmon resonance phenomenon can be used as a means for detecting or measuring sAPP3. A biosensor using the surface plasmon resonance phenomenon can observe the interaction between protein and protein as a surface plasmon resonance signal in real time using a small amount of protein and without labeling. (Eg, BIAcore; manufactured by Pharmacia). Therefore, it is possible to detect or measure sAPP / 3 by using a biosensor such as BIAcore.

That is, an antibody for detecting sAPP3 is brought into contact with a sensor chip on which a sample containing sAPP / 3 is immobilized, and the antibody that binds to the immobilized sAPP ^ is detected as a change in resonance signal. Is what you do. Specifically, it may be performed as follows. First, activate the sensor chip CM5 (manufactured by Biosensor) to immobilize sAPP / 3 in the culture supernatant on the sensor chip. That is, EDC I NHS aqueous solution (200 mM EDC (N- After activating the sensor chip with chill-N '-(3-dimethylaminopropyl) carbonate hydrochloride) and 50 mM NHS (N-hydroAPPysuccinimide), HBS buffer (10 mM HEPES ρΗ7.4, Wash the sensor chip with 150mM NaCl, 3.4m EDTA, 0.05¾Tween20).

 Next, an appropriate amount of an antibody specifically recognizing sAPP / 3 dissolved in HBS buffer is brought into contact with the sensor chip and immobilized. After washing the sensor chip with HBS buffer, the remaining active groups on the sensor chip are blocked with an ethanolamine solution (1M ethanol monoamine hydrochloride, pH 8.5). Wash the sensor chip again with HBS buffer.

 Next, an appropriate amount of antibody specifically recognizing sAPP / 3 dissolved in HBS buffer is injected. At this time, the amount of the antibody specifically recognizing sAPP3 bound to sAPP / 3 immobilized on the sensor chip is observed as an increase in the resonance signal value. At this time, a control group may be installed. As the control group, a negative control group containing no sAPP3, a positive control group containing sAPP3, or both groups can be used.

 The anti-sAPP / 3 antibody bound to sAPP / 3 bound to the sensor chip can be quantitatively measured as a change in resonance signal value. In this case, sAPPS is detected and determined by comparing the results obtained with the negative control group containing no sAPP3 and the results obtained with the positive control group containing no or sAPP / 3. be able to.

 In the detection or measurement of SAPP, sAPPS can be labeled and the label can be detected or measured as a means for detecting or measuring the bound protein.

For example, in the detection or measurement of sAPP3, an antibody that specifically recognizes SAPP was labeled in advance and incubated with sAPPS. Thereafter, the antibody is washed and specifically recognizes the bound sAPP / S, and the antibody is detected or measured by the label.

An antibody that specifically recognizes sAPP3 can be labeled by a generally known method. Examples of the labeling substance include a radioisotope, an enzyme, a fluorescent substance, and biotin-Z-avidin. As these labeling substances, commercially available labeling substances can be used. Is to radioisotopes, for example ^{3 2 P, 3 3 P,} 1 3 1 I, 1 2 5 I, 3 H, '4 C, 3 5 S and the like. Examples of the enzyme include alkaline phosphatase, phosphosperoxidase, / 3-galactosidase, β-darcosidase and the like. As the fluorescent substance, for example, fluorescein isothiocyanate (FITC) and rhodamine are cited. These can be obtained commercially, and are labeled by a known method.

 Specifically, it can be performed as follows. That is, a solution containing sAPP is added to the plate and left overnight. After washing the plate, block it with, for example, BSA to prevent non-specific binding of the protein. After washing again, an antibody that specifically recognizes sAPP3 is added to the plate. At the same time, place a group without sAPP / 3 (negative control) and a group to which Z or known concentration of sAPP3 was added (positive control), and incubate them.

After incubation, wash and detect or measure bound antibody. For detection or measurement, in the case of a radioisotope, detection or measurement is performed by liquid scintillation. In the case of an enzyme, the substrate is added, and the enzymatic change of the substrate, for example, color development is detected or measured by an absorptiometer. In the case of fluorescent substances, detect or measure with a fluorometer. By comparing these results with the values obtained for the control group, sAPP / 3 can be detected or measured. Specific for sAPP S as a means to detect or measure sAPP / 3 A primary antibody that specifically recognizes and a secondary antibody that specifically recognizes the primary antibody can be used.

 For example, an antibody specifically recognizing sAPP / 3 is brought into contact with a sample containing sAPP3, incubated, washed, and an antibody specifically recognizing sAPP / 3 bound thereto is obtained. It is detected or measured by a secondary antibody that specifically recognizes it. That is, preferably, an antibody that specifically recognizes sAPP3 is brought into contact with sAPP ^ bound to the support. After the incubation, the plate may be washed, and an antibody specifically recognizing bound sAPPyS may be detected or measured by a secondary antibody specifically recognizing the antibody. The secondary antibody is preferably labeled with a labeling substance.

 Specifically, it can be performed as follows. That is, a solution containing sAPP / 3 is added to the plate and left overnight. After washing the plate, block it with, for example, BSA to prevent nonspecific binding of the protein. Wash again and add an antibody that specifically recognizes sAPP / 3 to the plate. At the same time, place a group without SAPP (negative control) and a group to which Z or a known concentration of SAPP was added (positive control), and incubate them.

 After the incubation, wash and add a secondary antibody to the antibody that specifically recognizes sAPP / 3.After an appropriate incubation, wash to obtain a primary antibody that specifically recognizes the sAPP / 3. SAPP / 3 is detected or measured by a secondary antibody that specifically recognizes it. For detection or measurement, in the case of radioisotopes, detection or measurement should be performed by liquid scintillation. In the case of an enzyme, the substrate is added, and the enzymatic change of the substrate, for example, color development is detected or measured by an absorptiometer. In the case of a fluorescent substance, it is detected or measured by a fluorometer. By comparing these results with the values obtained in the control group, sAPP3 can be detected or Z-measured.

More particularly, the invention is particularly preferred for EL 1 SA (Enzyme-1inked). Immunosorbent Assay) can be performed as follows. In order to detect or measure SAPPS in the cell culture supernatant, for example, detection or measurement may be performed by a Sandwich-type ELISA. An antibody that specifically recognizes sAPP3 but does not recognize sAPPa is added to a 96-well immunoplate, and incubated at 4 ° C, and the antibody is coated on a plate. Next, the plate is washed with PBS (Phosphate Buffered Saline) or the like, and incubated with 25% block ace for 7 to 10 hours to perform blocking to prevent non-specific binding of proteins.

 Then, add a cell culture supernatant sample and a primary antibody that specifically recognizes the N-terminus of APP, and incubate at 4 ° C. After incubation, wash the plate with PBS and incubate with peroxidase-labeled secondary antibody for 1 hour at room temperature. After that, color is developed for 10 minutes at room temperature using a peroxidase substrate, and the absorbance is measured with a microplate reader (manufactured by Bio-Rat).

Detection or measurement of sAPP / 3 can also be used for High Throughput Screening (HTS). Specifically, by performing up to blocking manually, and then performing the subsequent reaction by robot, automating is performed, and high throughput screening can be realized. That is, phasing solid solutions containing sAPP 3 buffer one _{(0. 1 M NaHC 0 3,} 0.02¾ NaN 3, pH9.6) diluted with. Add an appropriate amount of the diluted aqueous solution to each well of a 96-well Imnoplate (manufactured by Nunc) and incubate at 4 ° C.

After washing each well three times with a washing buffer (prepared in PBS at 0.05¾ Tween20), add 5¾i BSA (manufactured by SIGMA) solution 201 in PBS and add the solution at 4 ° C. To lock. Next, set the blocked immu- noble plate in, for example, Biomek 2000 HTS systemCBeckman) and execute the system control program. This At this time, use Biomek 2000 dispenser (manufactured by Beckman) or Multipipette 96-well simultaneous dispenser (manufactured by Sagian) to dispense the solution into each hole of the immunoplate and remove the solution. It can be performed.

 In addition, EL404 microplate washer (Bio Tek) can be used for cleaning each hole of the immunoplate. In addition, a SPECTRAmaAPP250 plate reader (Molecular Devices) can be used for measuring the absorbance.

 The program is set to perform the following operations. That is, wash each well three times with a washing buffer, and dilute the antibody that specifically recognizes sAPP3 with a dilution buffer (1 BSA, 0.5SA Tween20, PBS). At the same time, place a group without SAPP (negative control) and a group to which known concentration of sAPP3 was added (positive control), and incubate them at room temperature for 1 hour.

 Wash each well three times with a washing buffer, add 5,000 heron anti-sAPP; 3 antiserum diluted 5,000-fold with a dilution buffer to each well, and incubate at room temperature for 1 hour. Wash each well three times with washing buffer, add 100 μl of alkaline phosphatase-labeled anti-rabbit IgG antibody (manufactured by TAG0) diluted 5000 times with dilution buffer to each well, and add 1 well at room temperature. Incubate for hours.

Each well is washed five times with washing buffer, color development solution (substrate of buffers _{over; 50 mM NaHC0 3, lOmM MgCl} 2, and dissolved concentration of 1 mg / ml in pH 9.8 p - two preparative Rofuenirufu Osufe bets ( Sigma))) was added to each well, and after reacting at room temperature, the absorbance at 405 nm was measured using a microplate reader, iomek pre- reader (Beckman / Molecular Devices). Measure using By comparing these results with the values obtained for the control group, sAPP; 5 can be detected or measured. Antibodies used to detect or measure sAPP / 3 are commercially available antibodies Alternatively, an antibody contained in a commercially available kit can be used, or it can be obtained as a monoclonal antibody or a polyclonal antibody using a known method.

 To obtain a polyclonal antibody, an animal is immunized with a sensitizing antigen, and after confirming that the desired antibody level in the serum of the animal has increased, the blood of the mammal sensitized with the antigen is removed. The serum is separated from the blood by a known method. A serum containing a polyclonal antibody may be used as the polyclonal antibody, and if necessary, a fraction containing the polyclonal antibody may be further isolated from the serum. .

 To obtain a monoclonal antibody, it is necessary to confirm that the level of the desired antibody is increased in the serum of a mammal sensitized with the above antigen, and then remove immune cells from the mammal and subject them to cell fusion. Good. In this way, a hybridoma producing the desired monoclonal antibody can be obtained.

 To obtain a monoclonal antibody from the hybridoma, a method of culturing the hybridoma according to a usual method and obtaining a culture supernatant thereof, or transferring the hybridoma to a mammal compatible therewith Transplanted and expanded to obtain ascites

The monoclonal antibody thus obtained can also be obtained as a recombinant antibody produced using a genetic recombination technique. Moreover, as long as it has a desired binding activity, it may be an antibody fragment or a modified antibody thereof. For example, antibody fragments include Fab, F (ab ') ₂ , Fv or single chain FV (scFV) in which Fv of H chain and L chain are linked by an appropriate linker. No.

The antibody obtained as described above can be separated from the host inside and outside the cell and from the host and purified to homogeneity. Separation and purification of antibodies used in the present invention It is sufficient to use the separation and purification methods used for ordinary proteins, and the method is not limited at all.

 For the concentration measurement or activity confirmation of the antibody obtained above, known methods, for example, ELISA, EIA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay) or fluorescent antibody method can be used. .

The primary antibody or secondary antibody obtained as described above can be labeled by a generally known method. Examples of the labeling substance include a radioisotope, an enzyme, and a fluorescent substance. As these labeling substances, commercially available labeling substances can be used. As a radioactive isotope, for example, ^{3 2 P, 3 3 P,} 1 3 1 I, 1 2 5 I, 3 H, '4 C, 3 5 S is cited et be. Examples of the enzyme include alkaline phosphatase, horseradish phosphate, / 3-galactosidase, and β-glucosidase. Examples of the fluorescent substance include fluorescein isothiosinate (FITC) and rhodamine. These are commercially available and are labeled by known methods ο

 The present invention provides (1) preparing DNA from the cell line of the present invention without stimulation, (2) preparing DNA from the cell line of the present invention under stimulation, and (3) preparing the DNA obtained in (1). Obtaining a DNA clone that is not present in the DNA and present in the DNA obtained in (2), and (4) isolating DNA encoding a protein having secretase activity. It relates to a method for cloning DNA encoding retase. The DNA prepared from the cells is preferably cDNA. The method for preparing cDNA may be in accordance with the method described herein.

For stimulation of the cell line of the present invention, a cell differentiation inducer can be used. As the cell differentiation inducing substance, preferably, phorbol ester, IL-3, retinoic acid and the like can be used. In order to obtain DNA clones that are not present in unstimulated DNA and are present in DNA prepared under stimulation, DNA clones prepared under stimulation and DNA prepared under non-stimulation must be used. What is necessary is just to remove the DNA that is already present. To remove the DNA present in both the stimulus-prepared and unstimulated-prepared DNA, use the differential roning method (Lau, F. et al., And Nathans, D.A. EMBO J. (1985) 4, 3145-3151), Diffraction-free spray method (Liang, P. and Pardee, AB Science (1992) 257, 967-971), Subtractive cloning method (Hedrick, SM et al., Nature (1984) 308, 149-153) or the serial analysis method (SAGE method) (Velculescu, VE et al., Science (1995) 270, 484- 487) etc. can be used.

 In the differential roning method, a cDNA library obtained under stimulation is labeled, a labeled cDNA library is introduced into a phage, and the phage is infected to cells, for example, E. coli. Sow the plate. The resulting plaque is transferred to two membranes, for example, a nitrocellulose membrane, and each membrane is hybridized with the cDNA obtained under no stimulation and the cDNA obtained under stimulation. CDNA can be isolated from plaques that specifically hybridize under stimulus and do not hybridize under no stimulus.

In the differential display method, a cDNA library obtained under no stimulation and a cDNA library obtained under stimulation are used as templates, and several kinds of oligo dT primers and primers are used. After performing a PCR reaction using a combination of several random primers, the PCR products generated from each are separated by gel electrophoresis. A detailed comparison of the pattern of appearance of the separated PCR products, i.e., bands, between unstimulated and stimulated, and increased under stimulated compared to unstimulated bands or unstimulated bands (Band signal intensity The band can be identified and isolated, and the full-length cDNA can be isolated from this DNA fragment.

 In the subtractive cloning method, cDNA obtained under no stimulation is labeled with biotin and the like, and the cDNA obtained under stimulation is hybridized. Next, the hybridized cDNA is removed with a corresponding label, for example, avidin. This may be repeated to isolate cDNAs that are specifically present under stimulation and not under unstimulated conditions. The cDNA clone thus obtained is introduced into appropriate cells, and the DNA encoding a protein having / 5-secretase activity is isolated according to the method for detecting or measuring sAPP3 described above. do it.

 The method described in the above reference can be used for the serial analysis method. The frequency of the gene expressed in unstimulated and stimulated cells, i.e., the mRNA abundance ratio, and the frequency of occurrence of the nucleotide sequence of each gene fragment by the serial expression method You can know. By comparing the frequency of occurrence of this gene between unstimulated and stimulated, we identified sequences that were not present under unstimulated condition but existed only under stimulated condition or sequences whose appearance frequency increased under stimulated condition compared to unstimulated condition. CDNA can be isolated based on

Example

 Hereinafter, the present invention will be described in more detail with reference to Examples, but the Examples do not limit the scope of the present invention.

 Example 1. Construction of APP expression vector

APP695 or the APP695 (APP695NL) cDNA introduced with the double mutation found in familial Alzheimer's disease was transferred to a mammalian cell expression vector PCEP 4 (Invitrogen, Calif.) An APP expression vector was created by incorporating it into a multi-cloning site.

 The APP695NL cDNA was prepared by using a Transformer Site-Directed Mutagenesis kit (Clontech) to convert the amino acid lysine at position 595 of APP695 cDNA into asparagine and 596 It was created by changing the amino acid methionine to leucine.

 Example 2. Creation of an APP expression clone

 Human megakaryoblastic cells are in RPMI 1640 containing 10% fetal serum, and human neuroblastoma SH-SY5Y cells are DME MZF-12 containing 10% fetal serum. Maintained inside. Megakaryoblastic cells used Lipofectamine (manufactured by Gibco), and SH-SY5Y cells used a calcium phosphate transfection system (manufactured by Gibco), and introduced an APP expression vector into the cells. Cells into which the gene had been introduced were selected using hygromycin B (manufactured by Wako Pure Chemical Industries, Ltd.) to obtain each expression cloned cell. The megakaryoblastic cell APP-expressing clones were named MKZA P8 and MK / A P9 cells. The megakaryocyte-based cell clones expressing APP containing the mutation were named MK / NL2 and MK / NL10 cells, respectively.

 Example 3. Eastern analysis

 Since APP is known to have a heparin binding site, secreted APP in the culture supernatant is first concentrated by a batch method using heparin-Sepharose (Pharmacia), and the sample is concentrated. Was used for Western analysis. That is, each culture supernatant was centrifuged at 1500 rpm for 5 minutes, and then incubated with heparin-Sepharose at 4 ° C for 2 hours.

After washing the Heparin-I Sepharose with PBS, the bound proteins were eluted using a 1.5 M NaCl / PBS solution. The eluted protein was added to a 2x sample buffer (70 mM Tris pH 6.7, 20% Equal amounts of glycerol, 5% 21-mercaptoethanol, and 2% SDS) were added, treated at 100 ° C for 5 minutes, and then subjected to SDS-acrylamide gel electrophoresis.

 After electrophoresis, blot on PVDF-Plus membrane (Funakoshi), react with primary antibody, incubate with each of the biotinylated secondary antibodies (Funakoshi), and perform Vectas tain elite kit. The primary antibody developed using 4-chloronaphthol (manufactured by Wako Pure Chemical Industries, Ltd.) as a substrate is a C-terminal 16 amino acid such as an anti-APPN-terminal antibody and sAPP. The 2570 antibody and the 2561 antibody, which are anti-sperm antisera obtained by using as antigens the anti-sperm antiserum and the sAPP3 C-terminal 7-amino acid as antigens, were used.

 The anti-sAPP / 3 antibody (2560 antibody, 2561 antibody) is a KLH (Keyhole) protein as a carrier protein at the N-terminal position of the C-terminal 7 amino acid of sAPPyS (GlulleSerGluValLysMet, SEQ ID NO: 3). Limpet hemocyanin) was fused, mixed with adjuvant, and then immunized with rabbits.

 In addition, the anti-sAPPa antibody (2570 antibody) is similar to the case of the anti-sAPP antibody, except that the C-terminal 16 amino acid of sAPP (Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys, SEQ ID NO: 4) ) Was combined with KLH and immunized to rabbits. It has been confirmed that the 2570 antibody, the 2560 and 2561 antibodies specifically recognize the sAPP and sAPP3 bands, which have slightly different molecular weights, and immunostain separately.

 Example 4. Western blot analysis of secreted APP

Megakaryoblastic cells and APP-expressing clones MKZAP8 and MK / AP9 cells were cultured in RPMI 1640 containing 10% fetal calf serum for 3 to 4 days. Figure 2 shows the results of Western analysis of the cultured culture supernatant. For each lane, about 600 1 of culture supernatant is used.

 As the primary antibody, the 2561 antibody (anti-sAPPyS) was used in lanes 1 to 3, the anti-APPN-terminal antibody was used in lanes 4 to 6, and the 2570 antibody (anti-sAPPa) was used in lanes 7 to 9.

 Lanes 1, 4, and 7 show the results of megakaryoblastic cells, lanes 2, 5, and 7 show the results of MKZAP8 cells, and lanes 3, 6, and 9 show the results of MK / AP9 cells. As is clear from FIG. 2, sAPP was not detected in megakaryoblastic cells, and a single band of sAPPa having a molecular weight of about 100 to 120 KDa was detected only in APP-expressing clone cells. On the other hand, sAPP yS was not detected in any cells.

 Example 5. Effect of phorbol ester

MKZA P8 Cells were 3Xl0 ⁵ pieces culture 6-well culture plates, phorbol - glycol ester (Phorbol 12-Myristate 13- Acetate, Sigma) was added 100 n M. The culture medium was changed every three days, and phorbol ester was allowed to act for six days. FIG. 3A shows the results of Western analysis of the culture supernatant on days 6, 12, 17, 21, and 24 of culture. For each lane, about 2501 culture supernatants were used. As the primary antibody, the 2561 antibody was used for lanes 1 to 5, and the 2570 antibody was used for lanes 6 to 10. The results on days 6, 12, 17, 21, and 24 of culture from the left lane are shown.

By the action of phorbol ester, sAPP ^ was detected simultaneously with sAPPa from day 12 of the culture. FIG. 3B shows the results of a similar experiment performed on APP / NL-expressing clone MKZN L2 cells and MKZN L10 cells. In this experiment, phorbol ester was allowed to act for 12 days. For each lane, about 125 1 of the culture supernatant was used. Lanes 1-2 used the 2561 antibody, and lanes 3-4 used the 2570 antibody. Each lane on the left shows the results for M KZN L 2 cells. MKZN Similarly, in the case of L cells, sAPP / 5 was detected by the action of phorbol ester, and the amount was higher than that in MKZA P 8 cells.

 Example ^ Enzymi Myno Assy

 The secretory APP in the culture supernatant of each cell was measured semiquantitatively by a sandwich-type Enzymimnoassay. That is, first, 96-well immunoplate (manufactured by Narken Nunc) was incubated with 2560 antibody or 2570 antibody (5000 nanograms Z-mol) at 4 ° C, and the antibody was co-incubated. One ting was performed. Next, the plate was washed with PBS and then blocked by incubating with a 25% proceeding plate for 7 to 10 hours.

 Then, each culture supernatant sample (40 /) and an anti-APP N-terminal antibody (final 500-fold dilution) were added and incubated at 4 ° C for 10 days. Further, the plate was washed with PBS, incubated with a peroxidase-labeled secondary antibody (manufactured by Biosource) for 1 hour at room temperature, and then treated with TMB Microwell Peroxidase-Xixy System (manufactured by Funakoshi). The color was developed for 10 minutes at room temperature, and the absorbance at 450 nm was measured using a microplate reader (manufactured by Bio-Latt). Antibodies 2560 and 2570 were purified using affinity columns (HyTrap, manufactured by Pharmacia) to which the C-terminal 5-amino acid of SAPP or sAPPa was bonded, respectively.

 Example 7. Quantification of secreted APP by Enzymimnoassay

The culture supernatant of APP-expressing SH-S Y5 Y / AP4 cells cultured in DME MZF-12 containing 10% fetal serum for 3 days was analyzed for the secretory APP enzyme. Figure 4 shows the results of the test. The culture supernatant was measured after dilution with DME MZF-12 containing 10% fetal calf serum. Both sAPPa and sAPP3 show an increase in absorbance from about 3000-fold dilution, and almost reached a plateau at concentrations over 100-fold dilution. However, it was found that each of these secretory APPs can be measured with high sensitivity using this assay system.

 Next, FIG. 5 shows the results of enzymatic immortalisation of the culture supernatant of MKZAP8 cells on which the phorbol ester used in FIG. 3 was acted. The 2570 antibody was measured using a sample diluted 100-fold with the culture solution, and the 2560 antibody was measured using a sample diluted 10-fold with the stock solution and the culture solution. Day 0 data are from the culture supernatant of MKZAP8 cells without phorbol ester. In Enzymimnoassay, sAPP yS was not detected in MK cells, but only sAPP was detected. Furthermore, sAPP3 was detected by the action of phorbol ester.

 APP was forcibly expressed in megakaryoblastic cells, which were originally known not to produce APP, and its metabolism was examined. In megakaryoblastic cells, APP was cleaved only by α-secretase, but not by α-secretase. This was clearly demonstrated by both Western analysis and Enzymimno assay.

 When ΑΡΡ is forcibly expressed in normal cells, ΑΡΡ is reported to be metabolized by both α-secretase and と -secretase, and in that sense, the cell line of the present invention is very unique. It is considered that

 The megakaryocyte cell line ΜΚ / ΑΡ8 was obtained on March 9, 1998 by the Institute of Biotechnology and Industrial Technology, Institute of Biotechnology (Tsukuba-Higashi 1-3-1-3, Ibaraki Prefecture) It has been deposited internationally under the Budapest Treaty under the deposit number FERM II-6287.

 Reference example

Since the enzyme used in this experiment was highly sensitive, this assay system and the cell line of the present invention in which ΑΡΡ was forcibly expressed were used. By using, it is possible to clone / 3—secretase. That is, a cDNA library is prepared using mRNA of human brain or SH-S Y5 Y cells which are thought to express 5-secretase.

 Use the pCEP4 vector as an expression vector in cells and incorporate the prepared DNA library into this vector. The prepared vector is introduced into the competent cells, and a pool of E.c01i containing about 50 clones per pool is prepared using a 96-well plate. The cell line of the present invention expressing APP is cultured in a 96-well plate, and DNA prepared from E.c01i of 2 to 4 pools is introduced.

 Perform sAPPyS enzyme assay on the supernatant of each cell culture 2-3 days later to detect positive pools. A clone of cDNA is prepared from the obtained pool of positive cDNA, and by repeating the same operation, cloning of 3-secretase can be advanced.

 In addition, since it was known that megakaryoblastic cells could be induced to differentiate by phorbol ester, it was examined whether or not phorbol ester alters the metabolism of APP in megakaryoblastic cells. As a result, it was found that treatment with phorbol ester resulted in the production of sAPP / 3 even in megakaryoblastic cells in which APP was forcibly expressed. Based on this fact, the cell line of the present invention can be used for cloning ^ -secretase. In other words, it is thought that 3-secretase was induced by phorbol ester, and it would be possible to proceed with cloning by differential display. Industrial applicability

By introducing the APP gene into cell lines, A cell line that does not express sex has been established. This cell line is — useful for isolating the secretase gene. In addition, using this cell line

/ S — Provides a method for cloning the secretase gene. Reference to Deposited Microorganisms under Rule 13bis of the Patent Cooperation Treaty and Depositary Institution

 Depositary name: Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology

 Address: Higashi 1-chome, Tsukuba, Ibaraki, Japan Microorganism (1) Display: MK / AP8

 Deposit number: FERM BP-6287

Deposit date: March 9, 1998

Sequence listing

SEQ ID NO: 1

Array length: 3 3 5 4

Sequence type: nucleic acid

Number of chains: double strand

 Topology: linear

Sequence type: c D N A

Array

 AGTTTCCTCG GCAGCGGTAG GCGAGAGCAC GCGGAGGAGC GTGCGCGGGG GCCCCGGGAG 60 ACGGCGGCGG TGGCGGCGCG GGCAGAGCAA GGACGCGGCG GATCCCACTC GCACAGCAGC 120 GCACTCGGTG CCCCGCGCAG GGTCGCG ATG CTG CCC GGT TTG GCA CTG CTC CTG

 Met Leu Pro Gly Leu Ala Leu Leu Leu

 Five

 CTG GCC GCC TGG ACG GCT CGG GCG CTG GAG GTA CCC ACT GAT GGT AAT 222 Leu Ala Ala Trp Thr Ala Arg Ala Leu Glu Val Pro Thr Asp Gly Asn 10 15 20 25

GCT GGC CTG CTG GCT GAA CCC CAG ATT GCC ATG TTC TGT GGC AGA CTG 270 Ala Gly Leu Leu Ala Glu Pro Gin lie Ala Met Phe Cys Gly Arg Leu

 30 35 40

 AAC ATG CAC ATG AAT GTC CAG AAT GGG AAG TGG GAT TCA GAT CCA TCA 318 Asn Met His Met Asn Val Gin Asn Gly Lys Trp Asp Ser Asp Pro Ser

 45 50 55

GGG ACC AAA ACC TGC ATT GAT ACC AAG GAA GGC ATC CTG CAG TAT TGC 366 Gly Thr Lys Thr Cys lie Asp Thr Lys Glu Gly lie Leu Gin Tyr Cys

60 65 70 9 S

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 082 SA2 LZ

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 99S 092 SS2 OSS

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 082 OZZ

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I0 ^ 10 / 66df / 13d n IS / 66 0AV AAG AAA CAG TAC ACA TCC ATT CAT CAT GGT GTG GTG GAG GTT GAC GCC 2142 Lys Lys Gin Tyr Thr Ser l ie His His Gly Val Val Glu Val Asp Ala 650 655 660 665

GCT GTC ACC CCA GAG GAG CGC CAC CTG TCC AAG ATG CAG CAG AAC GGC 2190 Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met Gin Gin Asn Gly

 670 675 680

 TAC GAA AAT CCA ACC TAC AAG TTC TTT GAG CAG ATG CAG AAC 2232 Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gin Met Gin Asn

 685 690 695

TAGACCCCCG CCACAGCAGC CTCTGAAGTT GGACAGCAAA ACCATTGCTT CACTACCCAT 2292 CGGTGTCCAT TTATAGAATA ATGTGGGAAG AAACAAACCC GTTTTATGAT TTACTCATTA 2352 TCGCCTTTTG ACAGCTGTGC TGTAACACAA GTAGATGCCT GAACTTGAAT TAATCCACAC 2412 ATCAGTAATG TATTCTATCT CTCTTTACAT TTTGGTCTCT ATACTACATT ATTAATGGGT 2472 TTTGTGTACT GTAAAGAATT TAGCTGTATC AAACTAGTGC ATGAATAGAT TCTCTCCTGA 2532 TTATTTATCA CATAGCCCCT TAGCCAGTTG TATATTATTC TTGTGGTTTG TGACCCAATT 2592 AAGTCCTACT TTACATATGC TTTAAGAATC GATGGGGGAT GCTTCATGTG AACGTGGGAG 2652 TTCAGCTGCT TCTCTTGCCT AAGTATTCCT TTCCTGATCA CTATGCATTT TAAAGTTAAA 2712 CATTTTTAAG TATTTCAGAT GCTTTAGAGA GATTTTTTTT CCATGACTGC ATTTTACTGT 2772 ACAGATTGCT GCTTCTGCTA TATTTGTGAT ATAGGAATTA AGAGGATACA CACGTTTGTT 2832 TCTTCGTGCC TGTTTTATGT GCACACATTA GGCATTGAGA CTTCAAGCTT TTCTTTTTTT 2892 GTCCACGTAT CTTTGGGTCT TTGATAAAGA AAAGAATCCC TGTTCATTGT AAGCACTTTT 2952 ACGGGGCGGG TGGGGAGGGG TGCTCTGCTG GTCTTCAATT ACCAAGAATT CTCCAAAACA 3012 ATTTTCTGCA GGATGATTGT ACAGAATCAT TGCTTATGAC ATGATCGCTT TCTACACTGT 3072 ATTACA TAAA TAAATTAAAT AAAATAACCC CGGGCAAGAC TTTTCTTTGA AGGATGACTA 3132 CAGACATTAA ATAATCGAAG TAATTTTGGG TGGGGAGAAG AGGCAGATTC AATTTTCTTT 3192 AACCAGTCTG AAGTTTCATT TATGATACAA AAGAAGATGA AAATGGAAGT GGCAATATAA 3252 GGGGATGAGG AAGGCATGCC TGGACAAACC CTTCTTTTAAAA GATGTGTCH CAATHGTAT 3312 AAAATGGTGT TTTCATGTAA ATAAATACAT TCTTGGAGGA GC 3354

Array length: 4 2

Sequence type: amino acid

 Topology: linear

Sequence type: Peptide

Array

 Asp Ala Glu Phe Arg Hi s Asp Ser Gly Tyr Glu Val Hi s Hi s Gin Lys

 5 10 15

 Leu Val Phe Phe Ala Glu Asp Val Gly Sern Lys Gly Ala l i e l ie

 20 25 30

Gly Leu Met Val Gly Gly Val Val lie Ala

 35 40

SEQ ID NO: 3

Array length: 7

Sequence type: amino acid

 Topology: linear

Sequence type: Peptide

Array

 Glu lie Ser Gl u Val Lys Met

 Five

SEQ ID NO: 4

Array length: 1 6

Sequence type: amino acid

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dsy Biv sq OJd na usy sA ¾iv UJQ 3JV nig ¾iv "10" 19 丄 "19 lOUO / 66df / X3d ZSZ.lS/66 OW Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn 545 550 555 560

Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr

 565 570 575

Thr Arg Pro Gly Ser Gly Leu Thr Asn ly Lys Thr Glu Glu He Ser

 580 585 590

 Glu Val Lys Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val

 595 600 605

 His Hi s Gin Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys

610 615 620

 Gly Ala lie He Gly Leu Met Val Gly Gly Val Val He Ala Thr Val 625 630 635 640 lie Val lie Thr Leu Val Met Leu Lys Lys Lys Gin Tyr Thr Ser lie

 645 650 655

His His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg

 660 665 670

 His Leu Ser Lys Met Gin Gin Asn Gly Tyr Glu Asn Pro Thr Tyr Lys

 675 680 685

 Phe Phe Glu Gin Met Gin Asn

 690 695

Claims

The scope of the claims

1. A cell line that produces amyloid precursor protein (APP), (2) expresses α-secretase activity, and (3) does not express 3-secretase activity under no stimulation.

 2. The cell line of claim 1, which expresses a detectable / S-secretase activity under stimulation.

 3. The cell line according to claim 1, which is an animal cell.

 4. The cell line according to any one of claims 1 to 3, which is derived from a human megakaryoblastic cell.

 5. The cell line of claim 4, deposited as FBRM BP-6287.

6. (1) introducing a DNA library into the cell line according to claim 1, (2) expressing the introduced DNA, and (3) selecting a cell expressing ^ -secretase activity, 4) isolating DNA encoding a protein having secretase activity from selected cells; and a method for cloning DNA encoding secretase.

 7. The cloning method according to claim 6, wherein the DNA library to be introduced is a cDNA library.

 8. The cloning method according to claim 7, wherein the cDNA library is a mammalian cell-derived cDNA library.

 9. The method of claim 8, wherein the cDNA library is a brain-derived cDNA library.

10. (1) DNA is prepared from the cell according to claim 1 without stimulation, (2) DNA is prepared from the cell according to claim 1 under stimulation, and (3) the DNA obtained in (1) is prepared. Obtaining a DNA clone that is not present in the obtained DNA and present in the DNA obtained in (2), and (4) encodes a protein having 3-secretase activity A method for cloning DNA encoding 3-secretase, comprising isolating DNA.

 11. The method of claim 10, wherein the DNA prepared from the cells is cDNA.

 1 2. A DNA encoding a protein having a / 5-secretase activity, obtained by the cloning method according to claim 6 or 10.