WO1999043702A1

WO1999043702A1 - PROTEIN BINDING TO NE-dlg

Info

Publication number: WO1999043702A1
Application number: PCT/JP1998/003740
Authority: WO
Inventors: Toshihiko Kishimoto; Shin-Ichiro Niwa; Hiroaki Kuwahara; Hideyuki Saya
Original assignee: Sumitomo Electric Industries, Ltd.
Priority date: 1998-02-25
Filing date: 1998-08-24
Publication date: 1999-09-02
Also published as: CA2322070A1; JPH11239483A

Abstract

A protein (named 'Nedasin') which binds to NE-dlg, shows no homology with any known mammalian protein and has a molecular weight of 51 kD; polynucleotides encoding the above Nedasin or its mutants; antisense polynucleotides comprising the complementary base sequences of the base sequences of the above polynucleotides; and an antibody recognizing the above-mentioned Nedasin or its mutants.

Description

Akitoda

Protein that binds to 1 g of NE-d

Technical field

The present invention relates to a protein that binds to NE-d1 g (including a splicing isoform thereof). The present invention also relates to a polynucleotide encoding the above protein. In addition, the present invention relates to a homolog of the protein or the polynucleotide. The present invention also relates to an antibody that recognizes the above-mentioned protein.

Background art

The d1g gene, a Drosophila tumor suppressor gene, is a gene that, when deleted, causes hyperplasia of adult disciogenesis. A group of genes having homology with the d1g gene is called MAGUK family, and is considered to have a function of localizing at cell adhesion sites and nerve endings and accumulating specific proteins. The present inventors isolated the NE-d1g gene, which is a human gene and has homology to the Drosophila d1g gene, and further isolated the NE-d1g gene encoded by the NE-d1g gene. — D 1 g protein (hereinafter abbreviated as NE—d 1 g), and revealed that it interacts with the C-terminal region of the APC tumor suppressor protein. Has a negative control on cell growth (Oncogene, 14, 2425-2433).

Disclosure of the invention

The present invention provides a protein that binds to the NE-d1g,

It is an object of the present invention to provide a polynucleotide to be loaded, and to provide an antibody that recognizes the protein.

The present invention provides a protein (named nedacin) which binds to 1 g of NE-d, has no homology to a known mammalian protein, and has a molecular weight of 5 lkD. Nedacin can be obtained by affinity-purifying the crude protein fraction of each tissue using a column on which 1 g of NE-d is immobilized. Further, the present invention provides a protein comprising the amino acid sequence described in any one of SEQ ID NOs: 1, 3, 5, and 7 in the sequence listing. These are nedacins (including sublysing isoformers) in humans.

The present invention also provides a protein comprising an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and binding to NE-d1 g. I do. It is a mutant of human nedacin S.

The present invention also provides a polynucleotide encoding the above-mentioned nedacin (including a mutant).

The present invention also provides an antisense polynucleotide consisting of a nucleotide sequence complementary to the nucleotide sequence of the polynucleotide.

Also, in the present application, a method for producing nedacin is disclosed. Specifically, a method is disclosed in which a transformant into which nedacin cDNA is introduced produces nedacin. Also disclosed is a recombinant nedacin produced by the method.

The present invention also provides an antisense polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of the polynucleotide encoding the nedacin or the polynucleotide encoding the nedacin mutant. The antisense polynucleotide is included in the polynucleotide, but in the present application, it is referred to as an antisense polynucleotide particularly when it is specified that the polynucleotide comprises a nucleotide sequence of an antisense strand. Representative Antisense Polynucleotides The present invention also provides polynucleotides that are all or part of a nucleotide consisting of 12 or more bases that encode nedacin or a nedacin variant.

This polynucleotide, for portions of the coding region, can be used to produce partial length proteins of nedasin or nedasin variants, respectively. Ma It can also be used as a probe.

The present invention also provides an antisense polynucleotide which is all or a part consisting of 12 or more bases of the antisense polynucleotide of nedacin or a nedacin mutant.

The antisense polynucleotide is capable of inhibiting the biosynthesis of nedacin or a nedacin mutant, respectively. It can also be used as a probe.

The present invention also provides a polynucleotide obtained by chemically modifying the above-mentioned polynucleotide (including an antisense polynucleotide).

In addition, the present application discloses that the analysis by the northern blot hybridization method using a DNA probe enables the detection of nedasin mRNA from each tissue and that the nedasin mRNA is naturally expressed. Is what you do. The present invention also provides a nedacin homolog protein encoded by a homolog gene of the nedacin gene. In the present application, when no derived animal name is indicated, nedacin in each vertebrate is collectively indicated. The above nedacin can be produced in a transformant into which the nedasin gene has been introduced. The present invention uses a polynucleotide encoding human nedasin or a part thereof consisting of at least 12 bases (a part of the coding region) as a probe to obtain nedasin from another vertebrate, preferably mammalian cDNA library. It also includes a method for obtaining cDNA and nedacin cDNA obtained by the method. The present invention also provides a nedacin protein which is a homologue of human nedacin encoded by the nedacin cDNA.

The present invention also provides an antibody that recognizes the nedacin and a mutant of the nedacin. In the present application, it is disclosed that the antigenicity of nedacin, that is, that an antibody can be produced from nedacin.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is a diagram showing the positional relationship between the cDNA of nedasin S, each fragment obtained during the cloning process, and each primer.

FIG. 2 is a diagram showing a positional relationship of each region of NE-d1g.

Fig. 3 is an electrophoresis photograph showing the result of SDS-PAGE electrophoresis of a protein extracted from E. coli brain cells binding to 1 g of NE-d and silver staining of the gel as it is. 1 is an electrophoretic photograph showing the result of electrophoresis of a protein extracted from a brain cell of a mouse bound to 1 g of a NE-d delivery mutant.

FIG. 4B is a diagram showing the relationship between the partial or full length of the NE-dlg delivery mutant.

FIG. 5 is an electrophoretic photograph showing the results of analyzing the expression of nedacin mRNA in each tissue by the Northern Protocol hybridization method.

FIG. 6 is an electrophoretic photograph showing the results of RT-PCR analysis of the expression of nedacin mRNA in each tissue and each cell.

FIG. 7 is an electrophoresis photograph showing the result of electrophoresis of an immunoprecipitate containing nedacin and 1 g of NE-d.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

(Purification of Nedacin)

The nedacin according to the present invention is obtained by combining a crude protein fraction extracted from cells derived from tissues of various animals as well as humans with 1 g of NE-d, and further dialysis, ammonium sulfate precipitation or gel filtration column, Purification can be performed singly by chromatography, using an exchange column or hydroxyapatite as an adsorbent, or by glycerol density gradient centrifugation. Nedacin can be purified by combining various types of chromatography. The purified nedacin fraction binds 1 g of NE-d.

The fraction containing purified nedacin was analyzed by SDS-polyacrylamide electrophoresis (S DS-PAGE ^ Ant ibodies A Laboratory Manu a 1, p636-640), and separation by two-dimensional electrophoresis using 0, Farrell, etc. Bands are observed.

(Isolation of nedacin)

Nedacin can be isolated from the band obtained by SDS-PAGE or two-dimensional electrophoresis by extracting a protein from a band having a molecular weight of about 51 kDa. That is, nedacin separated and identified by electrophoresis can be transferred to polyvinylidene difluoride (PVDF) or the like.

(Creating probes)

Nedacin after the above-mentioned treatment is subjected to various enzyme treatments such as lysylendopeptidase into a mixture of polypeptide fragments, and the obtained mixture of peptide fragments is separated by high performance liquid chromatography.

It is possible to determine the amino acid sequences of some of the obtained polypeptide fragments using an automatic amino acid sequencer or the like.

This can be achieved by selecting a polypeptide fragment encoding the amino acid sequence obtained by the above analysis, which has a low degeneracy frequency, and synthesizing and preparing a complementary polynucleotide corresponding thereto. . This synthetic polynucleotide can be used as a probe for searching for a polynucleotide encoding nedasin.

When a plurality of amino acid sequences are obtained, it is possible to combine the corresponding complementary polynucleotides, convert the cDNA into type III, and obtain a longer fragment DNA probe by PCR.

As described above, a polynucleotide probe for screening a polynucleotide encoding nedasin is obtained.

(Preparation and screening of cDNA library) For preparing a cDNA library for screening the polynucleotide encoding nedasin of the present invention, a general method can be used, for example, by the following steps.

That is, (i) separating messenger RNA (mRNA) from cells, (ii) synthesizing a single-stranded complementary polynucleotide (cDNA) and then a double-stranded polynucleotide from the mRNA,

(iii) incorporating the double-stranded polynucleotide into a plasmid or phage, (iV) transforming a suitable host cell with the obtained recombinant plasmid or phage,

(V) After culturing the obtained transformant, a plasmid or a plasmid containing the target polynucleotide is obtained from the transformant by an appropriate method, for example, colony hybridization or plaque hybridization. Isolating di,

(Vi) cutting out the target polynucleotide from the plasmid or phage,

(Vii) This is the step of subcloning the excised polynucleotide (cloned polynucleotide) into a suitable plasmid.

Each step will be described in more detail.

Step (i): mRNA encoding the polypeptide of nedasin can be obtained from various animal tissues, organs, and producer cells, more specifically, from the brain, placenta, liver, kidney, and the like.

In addition, as a method for preparing total RNA from the cells, guanidium / cesium chloride, a method (Guanidi umu / Cesi umu Chloride method, Mo lecular Clonin Second Edition, Cold Spring Harbor Laboratory or Pre- ss, p7. 19-7.23, 1989) Guanidimuthiocinate method (Analyt i cal Bio chemi stry, 162, pl 56—159, 1987) is generally used.

Separation and purification of mRNA from total RNA obtained by the above operation can be performed, for example, by using oligo dT-cellulose (Collaborative Research Co., Ltd.) or Oligodex-1 dT 30 (Yukara Co., Ltd.). This can be carried out by an adsorption column method or a batch method.

Step (ii): Using the mRNA obtained in this manner as type I and using reverse transcriptase, for example, the Okayama-Berg method (Okayama, H. and Ber, P., Molecular and Cereal) l lu l ar B ioloy, 3, p 280, 1983) cDNASynthesize cDNA according to the method of Gubler and V. Hoffman (Gubler, V. and Hoffman, BJ, Gene, 25, p 263-269, 1983) I do.

Step (ii): Incorporate the obtained cDNA into Plasmid II phage to prepare a cDNA library.

Examples of plasmid vectors incorporating cDNA include PBR322 (Gene, 2, 95, 1977), PBR325 (Gene, 4, 121, 1978), PUC12 or PUC13 (Gene, 19, 259, 1982), PUC 18 or PUC 19 (Gene, 33, 103, 1985), PU C 118 or PUC 119 (Me t ho ds in Enzymology, 153, 3, 1987), Blu scr ip t II (Nucleic Acids. Res., 17, 9494, 1989), etc., but any other one can be used as long as it is replicated and maintained in the host. .

Examples of the phage vector incorporating cDNA include, for example, Agt10 (Huynh, TV, Young, RA and Davis, .W., DNA cloning, A Practi ca l App roach, IRL Pre ss, Oxford, 1, 49, 1985), λ gt 11 (Proc. Natl. Ac ad. Sci., USA, 80, 1194, 1983) or human ZAP II (Nucleic Acids. Res., 17, 9494,

1989) can be used, but other vectors may be used as long as they can be propagated in an appropriate host.

Examples of a method for incorporating cDNA into plasmid include, for example, the method of Sambrook (J.), et al. (Mo 1 ecu 1 ar Cloning Sect on Ed Ed., Pi. 53— 1.73, 1989). As a method of incorporating cDNA into a phage vector, for example, the method of Hyunh, T.V., etc. (DNA cloning as described above) can be used.

Step (iv): Plasmid or phage vector obtained by the above-mentioned method is used in a suitable host such as Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis). And Saccharomyces cerevisiae (Saccharomyces escer evisiae) and the like, and can be transformed.

Methods for transforming a host with a plasmid vector include, for example, the electroporation method described in Molecular Cloning (Moe ecu 1 ar Cloning, supra, page 1.74-1.84) or Calcium chloride method and the like can be mentioned. In addition, a phage vector can be introduced, for example, into grown Escherichia coli using an in vitro packaging method.

Step (V): To select the desired cDNA of nedacin from the cDNA obtained by the above method, for example, a colony hybridization method using a labeled probe or a plaque hybridization method (see above) Mo 1 ecu 1 ar C loning S e c tion Ed it., 1.85— 1.104 or 2.112-2. 120) can be used.

Polynucleotide used as a probe in the above hybridization Any kind of polynucleotide can be used as long as it is a polynucleotide that hybridizes with nedacin. For example, a polynucleotide chemically synthesized based on the amino acid sequence of nedacin can be used.

As described above, a polynucleotide encoding nedacin can be prepared. (Determination of base sequence)

Determination of the nucleotide sequence of the cDNA obtained in accordance with the above can be performed by, for example, the Maxiam-Gilbert method (Methods in Enzymology, 65, 499-560, 1980), the dideoxy method (Messing, J. eta 1., Nucleic Acids Research, 9, 309, 1981), and Taq cycle sequencing using fluorescent dyes (Biotechniques, 7, 494-499, 1989). . If the sequence shows that DN 含む (hereinafter sometimes referred to as full-length DNA) containing the entire coding region could not be obtained, a combination of DNAs was selected to cover the coding region, By cutting and joining the DNA with an appropriate restriction enzyme site contained in the overlapping portion of the DNA, the full-length DNA can be obtained. Further, a primer is designed so as to include the entire coding region, and PCR is performed using the primers with a cDNA library of a target vertebrate, preferably a mammal, as a type I, to obtain a full-length primer. DNA can also be obtained.

(Human nedacin)

The determined human nedacin has four splicing isoforms. Nedacin S having an SSV structure at the C-terminus has a binding property to NE-d 1 g. As described in SEQ ID NO: 2 in the sequence listing, the nucleotide sequence of cDNA encoding nedasin S consists of 2040 residues, and the open reading frame is composed of 1365 bases from A at 86 to A at 1450. Yes, encoding 454 amino acids. From the structural point of view that it binds to NE-d 1 g, the C-terminus becomes an S SV structure. It is that it is wearing.

The nucleotide sequence of the cDNA encoding nedasin V1 consists of 1926 residues, as shown in SEQ ID NO: 4 in the sequence listing, and the open reading frame is from A at position 1 to A at position 16 Up to 1416 bases and encodes 471 amino acids.

As described in SEQ ID NO: 6 in the sequence listing, the nucleotide sequence of cDNA coding for nedacin V2 has an open reading frame consisting of 151 and 18 residues and encodes 505 amino acids.

As described in SEQ ID NO: 8 in the sequence listing, the nucleotide sequence of cDNA encoding nedacin V3 has an open reading frame consisting of 133 residues and encodes 460 amino acids. I do.

The c DNA according to the present invention includes c DNA consisting of the nucleotide sequence ATG is not bound to the 5 ⁵ end of the open reading frame of the sequence.

Not only cDNA, but also a DNA having a primary structure of the base sequence described in SEQ ID NO: 2, 4, 6, or 8 in the sequence listing, and an RNA comprising a base sequence corresponding to the base sequence, the human nedasin of the present invention. Included in the encoding polynucleotide.

The polynucleotides of the present invention also include DNAs that include 5'-flanking polynucleotides that encode part or all of the signal peptide for human nedacin. Furthermore, in accordance with the degeneracy of the genetic code, substituting at least one base of the polynucleotide for another type of base without changing the amino acid sequence of the polypeptide produced from the polynucleotide. Can be.

Therefore, the polynucleotide of the present invention also has an amino acid sequence represented by SEQ ID NO: 1, 3, 5, or 7 in the sequence listing by substitution based on the degeneracy of the genetic code. Includes everything that is

The amino acid sequence of human nedacin S of the present invention is the amino acid sequence described in SEQ ID NO: 1 in the sequence listing. The calculated molecular weight of the protein consisting of the amino acid sequence is 51.0. kD and the isoelectric point is 5.35.

The amino acid sequence of human nedacin V1 of the present invention is the amino acid sequence described in SEQ ID NO: 3 in the sequence listing. The calculated molecular weight of the protein consisting of the amino acid sequence is 52.8 kD, and the isoelectric point is 5.48.

The amino acid sequence of human nedacin V2 of the present invention is the amino acid sequence described in SEQ ID NO: 5 in the sequence listing. The calculated molecular weight of the protein consisting of the amino acid sequence is 56.8 kD, and the isoelectric point is 5.36.

The amino acid sequence of human nedacin V3 of the present invention is the amino acid sequence described in SEQ ID NO: 7 in the sequence listing. The calculated molecular weight of the protein consisting of the amino acid sequence is 52. lkD, and the isoelectric point is 5.90.

Human nedacin according to the present invention includes a polypeptide in which methionine is not bound to the N-terminal of the amino acid sequence. Also, an intermediate in which a part or all of a signal peptide for human nedascin is bound or deleted at the N-terminus of the amino acid sequence is included.

In addition, it is possible to change a part of the structure of a DNA encoding a protein without changing the main activity of the polypeptide by natural mutation or artificial mutation. Examples of artificial mutation include point mutation technology. Utilizing this technique, a DNA mutant is produced in which the structure of the DNA encoding the target protein is changed, and the DNA mutant is introduced into an appropriate host to prepare a transformant. A transformant can produce a mutant of the desired protein. Utilizing this technique, it is possible to produce a modification of the nedacin of the present invention in which the structure is changed. Therefore, the present invention relates to a mutant of nedacin S, which includes a protein in which the C-terminal S SV structure of the amino acid sequence has been changed while being conserved, and which binds to NE-d1 g. Mutants of the splicing isoforms nedacin V1, nedacin V2 or nedacin V3 are also included. In the case of producing a mutant by the technique of P70 site directed mugenesis, usually several amino acids can be substituted, deleted or added by one operation. By repeating this operation a plurality of times, more amino acids can be substituted, deleted or added, and a desired mutant can be produced. The homology between nedacin S of the present invention and its mutant is preferably 75% or more at the amino acid level, more preferably 90% or more, and even more preferably 95% or more.

The present invention also includes a polynucleotide consisting of a nucleotide sequence encoding a nedacin mutant.

CDNA and genomic DNA encoding nedasin can be used as they are or after being cut with restriction enzymes, depending on the purpose.

(Antisense polynucleotide)

The present invention also includes an antisense polynucleotide comprising a base sequence complementary to the base sequence. The antisense polynucleotide of the present invention can be used to inhibit the biosynthesis of human nedacin, and can also be used as a probe.

Generally, an antisense polynucleotide is included in a polynucleotide. In the present application, the antisense polynucleotide will be described as being included in the polynucleotide. However, when the antisense polynucleotide is specifically referred to as an antisense polynucleotide, it is referred to as an antisense polynucleotide.

The antisense polynucleotide for inhibiting the biosynthesis of the polypeptide preferably comprises 15 or more bases. On the other hand, antisense polynucleotides that are too long are unsuitable for uptake into cells. Generally, when the antisense polynucleotide is incorporated into cells to inhibit the biosynthesis of the target protein, the base is 12 to 30 bases, preferably 15 to 25 bases, and more preferably. Is an antisense polynucleotide comprising 18 to 22 bases It is better to use 7 The antisense polynucleotide to human nedacin of the present invention also comprises 12 to 30 bases, preferably 15 to 25 bases, more preferably 18 to 22 bases. It is preferable to use an antisense polynucleotide.

The antisense polynucleotide of the present invention includes all of those in which a plurality of nucleotides consisting of bases, phosphates, and sugars are bound, including those that are not naturally occurring. Typical examples are antisense DNA and antisense RNA.

For the antisense polynucleotide of the present invention, using known antisense technology, the binding strength to the target DNA or mRNA, tissue selectivity, cell permeability, nuclease resistance, or intracellular stability can be improved. High variety of antisense polynucleotide derivatives are obtained.

The derivatives generally known at present are preferably derivatives having at least one of enhanced nuclease resistance, tissue selectivity, cell permeability, and avidity. Particularly preferred are derivatives having a phosphorothioate bond as a skeletal structure. The polynucleotides and derivatives thereof of the present invention also include derivatives having these functions or structures.

In terms of ease of hybridization, it is generally said that an antisense polynucleotide or a derivative thereof having a base sequence complementary to the base sequence of the region forming the stem loop should be designed. The antisense polynucleotide and the derivative thereof of the present invention can form a stem loop as necessary.

An antisense polynucleotide having a sequence complementary to the sequence of the translation initiation codon, the ribosome binding site, the cabling site, and the splice site can generally be expected to have a high expression suppressing effect. Therefore, the antisense polynucleotide of the present invention or a derivative thereof, which is located near the translation initiation codon of DNA or mRNA encoding nedasin, a ribosome binding site, a caving site, Those containing a sequence complementary to the chair site are expected to have a high expression suppressing effect.

As long as it is a natural type antisense polynucleotide, the antisense polynucleotide of the present invention can be prepared by using a chemical synthesizer or by a PCR method using a gene encoding human nedacin as type III. In addition, some derivatives, such as a methylphosphonate type and a phosphorothioate type, can be chemically synthesized. In this case, the operation can be carried out in accordance with the instructions attached to the chemical synthesizer, and the resulting synthetic product can be purified by the HP LC method using reverse phase chromatography or the like. Antisense polynucleotide or a derivative thereof can be obtained.

(Polynucleotide probe)

Polynucleotide encoding nedacin of the present invention or a part thereof (polynucleotide comprising 9 or more consecutive bases), or an antisense polynucleotide of the antisense strand of the polynucleotide or a derivative thereof (from 9 or more consecutive bases) Antisense polynucleotide or a derivative thereof) can be used as a probe for screening a nedasin gene from a cDNA library or the like. At this time, those having a GC content of 30 to 70% can be suitably used. Further, a polynucleotide comprising 12 or more consecutive bases is more preferable, and a polynucleotide having 15 or more bases is more preferable. The polynucleotide used as a probe may be a derivative. Usually, a sequence having the number of bases or more is recognized as a sequence having specificity. As a cDNA library used in screening using the probe, a cDNA library prepared from mRNA can be preferably used. A group of cDNAs selected from these cDNA libraries by random sampling can be used as a sample for the search.

For example, DNA consisting of 9 or more consecutive bases in the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing or a polynucleotide that hybridizes to the DNA (antisense polynucleotide) is obtained by synthesizing the nedasin gene from a cDNA library or the like. It can be used as a probe for cleaning.

Further, by performing Northern blot hybridization analysis on mRNA derived from each tissue using the polynucleotide encoding the nedasin of the present invention or a part thereof as a probe, the tissue expressing nedasin-gene-derived mRNA can be identified. It is possible to find out.

(Chemically modified polynucleotide)

When chemically synthesizing DNA or RNA, chemistry such as methylation of the side chain or biotinylation, or substitution of S (sulfur) for 〇 (oxygen) in the phosphate group. Modification is well known.

In addition, it is also possible to label radioactive isotopes even with cDNA obtained from a cDNA library.

Therefore, the DNA and RNA of the present invention include the above-mentioned chemically modified DNA, RNA or antisense polynucleotide within its scope. The chemically modified DNA or RNA referred to here can exert both a protein-encoding function and a probe function, and the chemically-modified antisense polynucleotide can be used for probe or protein biosynthesis. It can exhibit both the function of inhibiting the activity and the function as a probe.

(Preparation of recombinant nedacin)

The present invention also includes a recombinant nedacin produced by the above-mentioned transformant into which the nedasin gene has been introduced. Further, the disclosure of the present invention makes it possible to produce a nedacin mutant in which one or more amino acids in the amino acid sequence of nedacin are substituted, deleted or added. As the vector and host used for the transformation, those described in the section on the preparation and screening of the cDNA library can be used. In addition, as the method for purifying nedacin or a nedacin mutant, the method described in the section on purification of nedasin can be used.

At the manufacturing stage, the nedacin or nedacin mutant to be produced is The transformant may be prepared as a fusion peptide with a peptide. In this case, in the purification step, it is necessary to carry out a treatment with a chemical substance such as bromocyan or an enzyme such as protease to excise nedacin or nedacin mutant.

(Antibody that recognizes nedacin)

Further, it is possible to prepare a polyclonal or monoclonal antibody against nedacin obtained in the present invention.

According to the present invention, an antibody recognizing nedasin can be obtained by immunizing a non-human animal with a peptide that is a part of nedasin of the present invention, as exemplified in Example 7, as to the antigenicity of nedasin Is to clarify. In addition, an antibody that recognizes nedacin can be obtained by immunizing with full-length nedacin. Therefore, an antibody recognizing nedacin of the present invention (hereinafter sometimes referred to as a human nedacin antibody) is an antibody obtained by immunizing a non-human animal with nedacin, and the antibody is the nedacin of the present invention. Antibodies whose recognition is confirmed by Western blotting, ELISA, immunostaining (eg, measurement by FACS), etc. are included in the range.

It is a commonly used method to use an immunogen obtained by binding a part of the protein to another carrier protein such as serum albumin. A part of the protein may be synthesized using, for example, a peptide synthesizer. In addition, it is preferable that a part of the protein has eight or more amino acid residues.

It is well known that if a polyclonal antibody can be obtained by immunization, a monoclonal antibody is produced by a hybridoma using lymphocytes of the immunized animal. (Chapter 6 of Ant ibodies A Lab or at Manly, mentioned above). Therefore, the nedacin antibody of the present invention includes a monoclonal antibody within its scope.

In the present invention, antibodies also include active fragments. An active fragment refers to a fragment of an antibody having antigen-antibody reaction activity. Specifically, F (ab ') ₂ , Fab', Fab, Fv and the like can be mentioned. For example, when the antibody of the present invention is digested with pepsin, F (ab,) ₂ is obtained, and when digested with papain, Fab is obtained. Reduction of F (ab,) ₂ with reagents such as 2-mercaptoethanol and alkylation with mono-acetic acid gives Fab '. Fv is a monovalent antibody active fragment in which a heavy chain variable region and a light chain variable region are linked by a linker.

A chimeric antibody can be obtained by retaining these active fragments and substituting the other parts with fragments of another animal.

The immunoassay using the obtained antibody provides a means for quantitatively measuring the change of the subject in various cells.

Specific examples of the method using an antibody include a method for detecting nedacin using a labeled nedacin antibody, and a method for detecting nedacin using a nedacin antibody and a labeled secondary antibody of the antibody. As the label, for example, a radioisotope (RI), an enzyme, avidin or biotin, or a fluorescent substance (FITC, rhodamine, etc.) is used.

Examples of the method utilizing an enzymatic reaction include an ELISA method, an immunoagglutination method, a Western blot method, a method for identifying an immune reaction molecule using flow cytometry, or a method similar thereto.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1> Isolation of NE—d1g binding protein

1. Preparation of GST—NE—d 1 g column

A vector containing the GST gene, between BamHI and HindII of pGEX-2TH (Pharmacia), consisting of the nucleotide sequence described in SEQ ID NO: 10 in the sequence listing. The base sequence up to the 1937th A was inserted, and the vector was introduced into Escherichia coli DH5 to prepare a transformant. The From the PDZ1 region consisting of the amino acid sequence from Ala at position 63 to G1u at position 568 of the NE-d1g protein (protein consisting of the amino acid sequence of SEQ ID NO: 9) in the transformant A GST-NE-d1g fusion protein in which GST was bound to the N-terminal of the portion up to the SH3 region was produced. Figure 2 shows the positional relationship of each area of NE-dig.

After immobilizing the GST-NE-d1g fusion protein on glutathione agarose beads, the beads were packed in a column to prepare an affinity column.

2. Affinity purification

The protein was extracted from P. cerevisiae brain cells, and the extracted crude fraction was applied to the affinity column at 4 ° C. and passed through the affinity column.

After the reaction, proteins elution buffer attached to the beads in a column (0. 5M NaC 1, 3 OmM T ris -HC 1 pH 7. 5, 1 mM EDTA, 5 mM M gCl 2, 1 mM DTT) pH 7 Washed out in 5 and heated in Laemli's sample buffer and placed at 100 ° C for 5 minutes.

The obtained sample was developed on SDS-PAGE and electrophoresed. As a result of silver staining of the gel, a protein with a molecular weight of 51 kD was isolated. The results are shown in Figure 3. Lane 1 is a lane in which only GST was electrophoresed, and lane 2 is a lane in which GST-NE-d 1 g fusion protein was electrophoresed.

3. Confirmation of binding with NE-d 1 g deletion mutant

In the same manner as above, a fusion protein was prepared by combining the following four types of NE-d 1 g of partial protein and full-length protein with GST, and an affinity column in which each protein was packed with beads was prepared. .

1: NE—A protein containing the dig SH3 region (amino acid sequence from Ser 503 to G1u at 568 in the amino acid sequence described in SEQ ID NO: 9 in the sequence listing).

2: full length NE—d 1 g. PT / JP98 / 03740

3: NE—Dig protein deleted from the GUK region (amino acid sequence from Arg at position 628 to Asp at position 803 in the amino acid sequence described in SEQ ID NO: 9 in the sequence listing).

4: NE-d 1 g protein with the N-terminal and GUK regions deleted.

FIG. 4 (B) shows the relationship between the part or the full length of NE—d 1 g of 1 to 4 above. To these columns, a crude protein fraction extracted from the E. coli brain cells was applied in the same manner as in 2 above, and the proteins bound to the fusion proteins immobilized on the beads were electrophoresed in the same manner as in 3 above. The result is shown in Fig. 4 (A). In FIG. 4 (A), the middle arrow indicates the protein band of 51 kD. This indicates that the 51 kD protein does not bind to the SH3 region, but binds to NE-d1 g lacking the N-terminal and NE-d1 g lacking the GUK region. . Therefore, it was found that the 51 kD protein binds to NE-dig and its PDZ region.

<Example 2> Determination of amino acid sequence of NE-d1g binding protein and nucleotide sequence of a gene encoding the same

1. Partial amino acid sequence

The gel which had been subjected to electrophoresis for the 51 kD protein purified in 2 of Example 1 was applied to a Immobilon-1 P VDF membrane (manufactured by Millipore) using a semi-dry electroprototyping apparatus, Sartoblot II-1 S ( (Manufactured by Sartorius).

The membrane filter on which the protein was blotted was washed with distilled water, stained with Ponceau S (P onceau S) staining solution (0.1% Ponceau-13, 1% acetic acid solution), and immersed in distilled water. The background was decolorized by shaking.

A spot at a position of 51 kD on this membrane was cut out and treated in situ with Lys-C protease (manufactured by Boehringer Mannheim) with an enzyme. The produced peptides were separated and purified by HP LC (Perkin Elmer) (flow rate 500 l / min, 0.1% TFA as solvent, 0-75% acetonitrile). Gradient, (// Bond Sphere C 8 pore size 300 Å (manufactured by Nihon Aid One Co., Ltd.) RP 300 column (2.

Some of the resulting polypeptide fragments of the 51 kD protein fragment were analyzed using a peptide sequencer (Prokin Sequencer-497 type analyzer manufactured by PerkinElmer).

By the above amino acid analysis, the following amino acid sequence was determined as a peptide fragment.

Peptide fragment 1: Va 1 Phe Leu G 1 u G 1 u A La Ser G in G in G 1 u Lys (from 43 to 53 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing) Array)

Peptide fragment 2: GluTrpCysPheLys (sequence from the 47th to the 51st amino acid sequence of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing) Peptide fragment 3: Thr Arg As LeuHislie Gin Ser H is (sequences 232 to 240 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing)

Peptide fragment 4: Asn Leu TyrPRoSeyrTyr Lys (sequences from 253 to 259 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing)

Peptide fragment 5: G1uPheAspAlaiLieLeuLieAsnProLys (sequence from position 394 to position 403 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing)

Peptide fragment 6: G1nVa1Va1ProPHeSer (sequence from amino acid position 446 to position 451 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing)

2. Sequence of base sequence of partial gene

(1) PCR EST database on the Internet based on the sequence of the peptide fragment (database in which the sequence of the mRNA fragment is registered, ht tp: //www.ncbi.nlm.nih.gov./dbEST ) The search resulted in selection of the sequence with accession number H 14396 and the sequence with accession number R 39820. A PCR primer was prepared based on the nucleotide sequence.

A human fetal brain cDNA library (Clontech) was used for PCR type II.

As a sense primer, P1 primer: 5'-ATGCCTGGGCTG GTTGATACACAC-3, (base sequence described in SEQ ID NO: 11), and as an antisense primer, P2 primer: 5'-CATCACTGTCT TATTTGTCAAAAG-3, The nucleotide sequence described in SEQ ID NO: 12 in the sequence listing was designed and synthesized using a DNA synthesizer (ABI, model 392). The synthesized primer was adjusted to 10 pmo1 / 〃1 with distilled water (the subsequent primer synthesis was performed in the same manner). When PCR was performed using these primers, a cDNA fragment of 609 bp was obtained. This fragment is hereinafter referred to as fragment A.

(2) Preparation of transformant

The fragment A obtained above was subjected to mini gel electrophoresis (0.75% agarose gel), and a band (609 bp) of the DNA was cut out from the gel. The cDNA was collected on a dialysis membrane, and the bands were checked by mini gel electrophoresis.

cDNA a l _y l up, was diluted with 99 1 of TE. The absorbance at 260 nm (A260) was measured, and the DNA concentration was calculated (the DNA concentration when the concentration of 260 was 1.0 was 50 l / ml). The DNA was diluted with TE so that the DNA concentration was.

(3) Determination of base sequence

Fragment A with P1 sense primer and P2 antisense primer Was used to perform a direct sequence. DNA sequencing was performed using the Auto Sequencer (ABI Model 373A) by the Dye-Mine-Mine-One-Duct method to determine the nucleotide sequence of fragment A cDNA.

3. Synthesis of primer

The following primers were synthesized to obtain further DNA fragments from the human cDNA library.

P 3 primer on the 3 ′ side from P 1 primer: 5′-TGATTTGCACA TTCAGAGCCATAT—3, (base sequence described in SEQ ID NO: 13 in the sequence listing), P 4 primer on the 5 ′ side from P 2 primer: 5 , -AGGTCTATGCTA CTTCCAGCAAAG-3, (base sequence described in SEQ ID NO: 14 in the sequence listing) was designed and synthesized using a DNA synthesizer (ABI, model 392). The synthesized primer was adjusted to 10 pmo1 / 1/1 with distilled water.

4. PCR

For the cDNA library, a cDNA library of human fetal brain (manufactured by Clonetech) was used, and the AP-1 primer set in the linker of the cDNA library: 5'-CCAT CCTAATACGACTCACTATAGGGC-3 ' One step PCR was carried out using P1, P2, P3 and P primers.

(1) 3, side cloning

The P1 primer was used to extend the single-stranded DNA on the 3 side by PCR.

c DNA 1.Ομ.1

dNTPmix (2.5 mM each) 1 2ul

P 1 Primer 1 0 1

3.3 XPCR buffer 45 jl

25mM Mg (0 A c) ₂ 0.

Distilled water 6 4 z 1 Total 14.7 1

Mineral oil 10〃1 is overlaid on the liquid having the above composition, left at 95 ° C. for 5 minutes, and XL Polymerase 0.3 / 1 (registered trademark, manufactured by Parkin Elma Co., Ltd.) was added. The cycle of “6 ° C for 30 seconds, 60 ° C for 30 seconds, and then 72 ° C for 2 minutes” was repeated 40 times. Finally, a fragment extension reaction was performed at 72 ° C for 7 minutes to complete the PCR.

After the reaction, the PCR product was transformed into type III and cloned on the 3 side to synthesize double-stranded DNA.

PCR product 0.5 / 1

dNTPmix (2.5 mM each) 1.2 μ. \

Ρ 3 primers 1.

ΑΡ— 1 Primer 1. 0 1

3.3 XPCR buffer 4.5 1

25mM Mg (OA c) ₂ 0.6 / 1

Distilled water 5.

Total 14.7〃 1

Mineral oil 10〃1 is overlaid on the liquid of the above composition, left at 95 ° C for 5 minutes,

Add XL Polymerase 0.3〃1 (registered trademark, manufactured by PerkinElmer Inc.) and add “9

A cycle of “6 ° C. for 30 seconds, 60 ° C. for 30 seconds, and then 72 ° C. for 2 minutes” was repeated 40 times. Finally, a fragment extension reaction was performed at 72 ° C for 7 minutes to complete PCR.

After the reaction, the PCR product was subjected to minigel electrophoresis on a 1.5% agarose gel. A fragment encoding a DNA fragment of about 1.2 kb was cut out and the PCR product was recovered. Further, the minigel electrophoresis was performed again on a part of the collected product, and it was confirmed that a band appeared at about 1.2 kb. Hereinafter, this DNA fragment is referred to as fragment B. (2) DNA sequence

1〃1 of the fragment B obtained above was taken and diluted with 99〃1 of TE. The absorbance at 26 Onm (A260) was measured and the DNA value was calculated (A260 value 1.0 was calculated as 50 µg / ml). A sample was prepared so that the DNA was 1 μg / m 1 based on the A260 value. P-3 primer and AP-1 Primer AP-2 primer set on the 3 'side: 5, -ACT CACTAT AG GGCTCGAGCGGC-3 (using the nucleotide sequence of SEQ ID NO: 16 in the sequence listing) The nucleotide sequence of fragment c was determined by the Dye-Minner-One-Yet method using an ABI AutoSynchencer-1 model 373S.

(3) 5, Side cloning

The P2 primer was used to extend the single-stranded DNA on the 3 side by PCR.

c DNA 1.0 / 1

dNTPmix (2.5 mM each) 1.2 μ. \

P 2 Primer 1.0 ζ 1

3.3 XPCR buffer 4.5 ζ 1

25mM Mg (0 A c) ₂ 0.6 1

Distilled water 6.4 6.1

Total 14.1 μ. \

A mineral oil (10-1) is overlaid on the liquid having the above composition, left at 95 ° C for 5 minutes, added with XL Polymerase 0.3-1 (registered trademark, manufactured by PerkinElmer Co., Ltd.), and added to “96 ° A cycle of 30 seconds at C, 30 seconds at 60 ° C, and then 2 minutes at 72 ° C "was repeated 40 times. Finally, a fragment extension reaction was performed at 72 ° C for 7 minutes to complete PCR.

After the reaction, the PCR product was transformed into type I and the 5 side was cloned to synthesize double-stranded DNA.

PCR product 0.5 1 dNTPmi x (2.5 mM each) 1.2〃1

P4 primer 1.0 1

AP-1 Primer 1.0 1.

3.3 XPCR buffer 4.5〃1

25mM Mg (〇Ac) ₂ 0.6 1

Distilled water 5.9 5.1

Total 14.7 1

Mineral oil 10〃1 is overlaid on the liquid of the above composition, left at 95 ° C for 5 minutes, and XL Polymerase 0.3〃1 (registered trademark, manufactured by PerkinElmer Inc.) is added. A cycle of “30 seconds, 60 seconds for 30 seconds, and then 72 ° C. for 2 minutes” was repeated 40 times. Finally, a fragment extension reaction was performed for 72 minutes at 72 to complete the PCR.

After the reaction, the PCR product was subjected to minigel electrophoresis on a 1.5% agarose gel. A fragment encoding a DNA fragment of about 0.5 kb was cut out and the PCR product was recovered. Further, the minigel electrophoresis was performed again on a part of the recovered product, and it was confirmed that a band appeared at about 0.5 kb. Hereinafter, this DNA fragment is referred to as fragment C hereinafter.

(4) DNA sequence

1〃1 of the fragment C obtained above was taken and diluted with 99〃1 of TE. The absorbance at 260 nm (A260) was measured, and the DNA value was calculated (A260 value 1.0 was calculated as 5 O ^ gZml). A sample was prepared such that the DNA had an A260 value of 1 μg / m 1. Using the P4 primer and the AP-2 primer, the nucleotide sequence of the fragment C was determined by the Dye-Mine-One method using an ABI AutoSynchencer model 373S.

5. Full-length cDNA conversion

Based on the base sequence of fragments A, B and C, P GCTCAGATGCCG-3 '(base sequence described in SEQ ID No. 17 in the sequence listing) was set as an antisense primer, P6 primer 1: 5, -AAAT AGGAT CC, the base sequence described in SEQ ID No. 18). PCR was performed under the following conditions using human fetal brain cDNA library as a type III.

dNTPmi 1.2 μΛ

MA 1.01

Mer 1.0 / 1

3.3 XPCR buffer 4.5 1

25mM Mg (OAc) 0.6 1

Distilled water5.4 1

Total 14.Ί μ. \

Mineral oil 15〃1 was overlaid on the liquid having the above composition, left at 96 ° C. for 5 minutes, and XL Polymerase 0.3〃1 (registered trademark, manufactured by PerkinElmer Inc.) was added. A cycle of “30 seconds, 60 seconds at 30 seconds, and then 72 ° C. for 1.5 minutes” was repeated 40 times. Finally, a fragment extension reaction was performed at 72 ° C for 7 minutes to complete PCR. This full-length cDNA was sequenced according to the method of dye and minerals. After confirming that the desired cDNA containing the full-length coding region was obtained, the cDNA was ligated to a protein that binds to NE-d1 g (this protein was nedasin (NEDAS IN, NE-DiAs soci ating protein IN). ) Was identified as cDNA encoding). This nucleotide sequence is shown as SEQ ID NO: 2 in the sequence listing. The gene consisting of this nucleotide sequence was named nedacin S gene. No known mammalian genes having homology with the nedacin S gene were found. Nedacin S cDNA and the position of each fragment and each primer obtained during the cloning process. Figure 1 shows the relationship.

The amino acid sequence of the nedacin S protein encoded by this gene (hereinafter sometimes simply referred to as nedacin S) is shown in SEQ ID NO: 1 in the sequence listing. The C-terminus of nedacin S was an S SV motif that could bind to the PDZ region. No mammalian protein having homology with this nedacin S was found, but it had about 40% homology with the yeast YDL238c protein.

6. Preparation of transformants

The full-length nedasin cDNA obtained above was inserted into a plasmid vector pGEM (registered trademark) — T Easy (Promega) multi-cloning site. This plasmid vector was introduced into E. coli NM522 to prepare a transformant. The Escherichia coli harboring the cDNA of nedacin S was named Ne dasin S, and the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry (1-3 1-3 Tsukuba East, Ibaraki, Japan (zip code 305- 8566)) and deposited on February 25, 1998. The accession number is FERM P-16663. Furthermore, on August 24, 1998, the Nedas inS was transferred to the Deposit under the Budapest Treaty at the institute (Accession No. FERM BP-6471).

<Example 3> Expression of nedacin in each tissue

For each of human poly A + RNA (mRNA) and human various cells poly A + RNA (mRNA), 2〃 g of each membrane (Huma n Mult ip le Ti s sue Northern Blot, II and III (manufactured by Clontech)), the labeled full-length nedasin S gene was added to the MoE ecu1arClonin A Laboratory Manual Manual, Edition Ed. According to the description, hybridization was carried out at 43 ° C. for 16 hours, and analysis was carried out by the Northern blot hybridization method.

'Gene labeling was performed by the following procedure. T

1) Labeling was performed using Takara MEGA LAB EL kit (registered trademark, manufactured by Takara Shuzo) according to the instruction manual.

2) Next, a DNA reaction solution having the following composition was prepared, and this solution was incubated at 37 ° C for 20 minutes, and then 25 1 TE was added to inactivate the enzyme.

DNA probe (2 pmo 1 / 〃 1) 14〃 1

10x phosphorylation buffer 2.5〃1

CNTP mixture (mix tur e) 2.5〃1

[Ar 32P] CLTP (37 OMBq / ml) (Amersham)

5 ^ 1

Klenow fragment

Total 25 1

3) Sephadex G-25 (registered trademark, manufactured by Pharmacia) equilibrated with TE 50 (5 OmM TrisC 1 pH 8.0, 1 mM EDTA) was applied to a 1.5 ml polyprep column (manufactured by BioRad). The DNA reaction solution heat-treated in 2) was packed on the column so that the bed volume became 1 ml.

4) Thereafter, 200 1 TE 50 was passed through the column four times, and a labeled DNA probe was obtained from the fraction eluted in the second 200-1.

FIG. 5 shows the results of analysis by the Northern Protocol hybridization method. From this, it was found that mRNA of nedacin was strongly expressed in brain, placenta, liver and kidney.

Example 4> Identification of Splicing Isoforms

MRNA was extracted from each tissue in which nedacin expression was observed using a Microfast Track (registered trademark) kit (manufactured by Invitrogen) mRNA purification kit, and RT-PCR method (new cell experimental engineering protocol, Shujunsha, Published in 1993, listed on pages 175-176). The obtained gene product was sequenced in the same manner as described above by the dye-mine method. As a result, three splicing Form was found to exist. These were named Nedacin VI, Nedasin V2, and Nedasin V3.

The amino acid sequence of nedacin V1 is shown in SEQ ID NO: 3 in the sequence listing, and the nucleotide sequence of the nedacin V1 gene is shown in SEQ ID NO: 4 in the sequence listing.

The amino acid sequence of nedacin V2 is shown in SEQ ID NO: 5 in the sequence listing, and the nucleotide sequence of the nedacin V2 gene (only the coding region) is shown in SEQ ID NO: 6 in the sequence listing.

The amino acid sequence of nedacin V3 is shown in SEQ ID NO: 7 in the sequence listing, and the nucleotide sequence of the nedacin V3 gene (only the coding region) is shown in SEQ ID NO: 8 in the sequence listing.

The nedacin VI gene was introduced into a plasmid vector pGEM (registered trademark) -TEASy (promega) multi-cloning site. The transformant in which this plasmid was introduced into Escherichia coli DH5 was named Ne dasin VI. Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry (1-3 1-3 Tsukuba East, Ibaraki, Japan) (Postal code 305-8566)) on February 25, 1998. The accession number is FERM P-16664. In addition, on August 24, 1998, the Nadasin V1 was transferred to the Deposit under the Budapest Treaty at the same institute (Accession No. FERM BP-6472).

RT_PCR was also performed on each cell to examine the expression of the nedasin splicing isoform.

FIG. 6 shows the results of RT-PCR for each tissue and each cell. This confirmed that nedacin S is predominantly expressed in human fetal brain and placenta, and nedacin V1 is predominantly expressed in other tissues.

<Example 5> Confirmation of binding between NE-dig and nedacin

1. Immunoprecipitation

The full-length nedasin S cDNA and nedasin V1 cDNA were obtained by inserting a 63-mer linker containing the my c tag into the EcoRI-BamHI site of the plasmid vector pBj-1. It was inserted into the BamHI site of my c. Each of these was introduced into COS cells together with a vector-pCGN (having an HA tag) incorporating the NE-d1 g gene in which the GUK region had been deleted, to prepare transformants.

After culturing this transformant, the cells are lysed using TNN buffer (15 OmM NaCl, 5 OmM Tris-HC1H7.5, 0.5% NP-40), and the cells are extracted. A liquid was obtained.

An anti-myc antibody was added to the cell extract, and the mixture was centrifuged at 3000 rpm for 5 minutes to separate immunoprecipitates.

2. Western blot

This immunoprecipitate was electrophoresed on 9% SDS-PAGE.

After electrophoresis, the DNA was transferred to a nitrocellulose membrane using a Transplot system (manufactured by Marisol).

The nitrocellulose membrane was immersed in 10% skim milk / PBS, 0.1% Tween 20, allowed to stand for 1 hour, and blocked. Thereafter, the plate was washed twice with 0.3% Tween20 / PBS for 5 minutes each.

After the anti-myc antibody was diluted to 1 g / ml with PBS, it was added to a nitrocellulose membrane and reacted at room temperature for 60 minutes. Thereafter, the plate was washed three times for 5 minutes each with 0.3% Tween20 / PBS.

Next, a solution obtained by diluting a peroxidase-labeled anti-mouse IgG (manufactured by Amersham) by 20000 times with PBS was added to the nitrocellulose membrane, and the mixture was further reacted at room temperature for 40 minutes. Thereafter, the plate was washed three times for 5 minutes each with 0.3% Tween 20 / PBS.

Next, 5 ml of a color developing solution of an ECL kit (manufactured by Amersham) was added to the nitrocellulose membrane, and reacted for 1 minute. Then, the membrane was exposed to X-ray film for 20 seconds, developed, and photographed. Fig. 7 shows the results.

As a result, nedacin S whose C-terminal is S SV binds to NE-d 1 g, but C-terminal However, it was found that nedacin V1 of PFP did not bind to NE-d1g. In other words, the binding between nedacin and NE-d1 g was demonstrated in vivo, and the binding was found to be controlled by a change in the C-terminal side of nedacin.

<Example 6> Chromosome mapping

Chromosomal mating of the full-length gene of nedacin S was performed as follows.

1. PCR

The PCR uses the Genebridge 4 Radiation Hybrid Screening Panel (available from Research Genetics, Inc.) as a type II to convert a portion of the Nedacin S gene. Primers: S1 primer (sense direction): 5, ATTGAAGAGGTTTATGTGTTC-3, (base sequence described in SEQ ID NO: 19 in Sequence Listing) and AS1 primer (antisense direction): 5 , -CAAGGGAGATGCACAACCACGCTA-3, (the base sequence described in SEQ ID NO: 20 in the sequence listing) were synthesized by a DNA synthesizer, and dissolved in distilled water to give 10 pmo \ / UL1.

The PCR operation was performed as follows. A liquid having the following composition was placed in a test tube and left at 96 ° C for 9 minutes.

Kit panel 1 1

dNTP mixture 1.5 1

S 1 Primer

AS 1 primer 1/1

10 times concentration of PCR buffer 1.5 1

25mM M g C 1 = 1.1

Distilled water 7.4 // 1

Ampli Taq Gold (registered trademark, manufactured by Pakinkin Elma)

0.1 μ.1 Total 15/1

Thereafter, the cycle of “96 ° C. for 30 seconds, followed by 54 ° C. for 30 seconds, and then 72 ° C. for 15 seconds” was repeated 35 times to carry out the reaction. Finally, the reaction was carried out at 72 ° C for 7 minutes to extend the fragment, thereby completing the PCR operation. Thereafter, 5-1 of the PCR product was subjected to 2.5% agarose gel electrophoresis.

2. Mapping

As a result, the section number of the panel to which the primer (S1 or AS1) was hybridized was changed to the “Whitehead Insitute / MIT C enterf or Genome Research (http: www—genome.wi.mit.edu/cgi”) — E-mail to b in / cont ig / rhmap er. Pi.;) To obtain information on the chromosome position and nucleotide sequence of the panel with the number. As a result, the nedacin S gene was located on the short arm of chromosome 9.

<Example 7> Preparation of antibody recognizing nedacin

1. Preparation of antigen

Based on the amino acid sequence of nedacin, the following peptides were synthesized using a peptide synthesizer.

Peptide A: RNIEEVYVGGKQVVPFSSSV (amino acid sequence from 435 to 454 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing) Peptide B: LYP S YKNYT S VYDKNNLLT (position 254 in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing) Amino acid sequence from position 272 to position 272)

A reaction solution was prepared by binding 2 mg of the prepared peptide to 2 mg of maleimide KLH (Pierce). The reaction was performed according to the method described in the instruction manual of Pierce.

2. Immunity

The antigen solution containing the peptide eight and peptide B (1 μg / ml) 100/1, 0.5 ml of PBS and 0.5 ml of Freund's complete adjuvant (manufactured by Difco) were taken in a syringe, mixed to give an emulsion, and subcutaneously inoculated into four parts on the back of a perch.

One week later, a second immunization was performed. From the second time on, immunization was performed by changing the adjuvant to Freund's complete adjuvant (Difco). Other operations are the same as the first operation. The second and subsequent immunizations were performed at weekly intervals for a total of six immunizations.

3. Antibody purification

One week after the final immunization, blood was collected. The blood was allowed to stand at room temperature for 3 hours to sufficiently coagulate the blood, followed by centrifugation at 3,000 rpm for 5 minutes, and the supernatant (serum) was collected.

Saturated ammonium sulfate was added to this serum to a final concentration of 50%, followed by salt prayer. This sample was centrifuged to precipitate a fraction containing the antibody. After that, the precipitate was dissolved in PBS and further salted out to PBS.

Thereafter, the antibody was affinity purified using a protein G Sepharose column (registered trademark, manufactured by Pharmacia). As a result, a total of 5 mg of the peptide-specific antibody was obtained.

Industrial applicability

Nedacin (including splicing isoform) or a mutant thereof of the present invention is effective for analyzing the mechanism of NE-d1 g.

The polynucleotide encoding the nedacin or a mutant thereof of the present invention, a polynucleotide consisting of a continuous base sequence of 12 or more bases, or a polynucleotide (antisense polynucleotide) that hybridizes to the polynucleotide, is a cDNA. It can be used as a probe for screening nedacin or its mutant gene from a library or the like.

In addition, the antisense polynucleotide of the present invention is characterized in that nedacin or a mutant thereof Biosynthesis can be inhibited.

The antibody of the present invention can be used for elucidating functions such as expression of nedacin and elucidating the formation mechanism of malignant tumors.

Claims

The scope of the claims

1. NE—a 51 kD protein that binds to 1 g of d.

2. A protein comprising the amino acid sequence of SEQ ID NO: 1, 3, 5 or 7 in the sequence listing.

3. A protein comprising an amino acid sequence in which one or more amino acids have been substituted, deleted or added in the amino acid sequence of SEQ ID NO: 1 in the sequence listing, and which binds to NE-d1g.

4. A polynucleotide encoding the protein according to any one of claims 1 to 3.

5. A part of the polynucleotide according to claim 4, wherein the polynucleotide comprises 12 or more consecutive bases.

6. An antisense polynucleotide comprising the nucleotide sequence of the antisense strand of the polynucleotide according to claim 4, or a part of a derivative of the antisense polynucleotide, wherein the polynucleotide comprises 12 or more consecutive bases. .

7. The polynucleotide according to any one of claims 4 to 6, which is chemically modified.

8. A method for obtaining cDNA which is a homolog of DNA consisting of the nucleotide sequence of SEQ ID NO: 2, 4, 6, or 8 in the sequence listing, wherein the cDNA according to any one of claims 5 to 7 A method in which a polynucleotide comprising the coding region of a polynucleotide is used as a probe, and a cDNA that hybridizes with the polynucleotide used as the probe is obtained from a cDNA library.

9. Consists of the nucleotide sequence of SEQ ID NO: 2, 4, 6 or 8 in the sequence listing

A cDNA which is a homolog of DNA and which is obtained by the method according to claim 8.

10. A homolog of the protein according to claim 2, wherein the protein comprises the amino acid sequence encoded by the cDNA according to claim 9. An antibody that recognizes the protein according to claim 1, 2, 3, or 10.