JPH11151096A - Protein having neurocyte function-activating activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new protein derived from human cerebrocortex for treatment, etc., of neurodegenerative diseases such as ischemic encephalopathy, Alzheimer's disease and Parkinson's disease, composed of a protein comprising a specific amino acid sequence and having neurocyte function-activating activity. SOLUTION: This new protein HUCEP-14 comprises a protein comprising an amino acid sequence represented by the formula or a protein comprising an amino acid sequence in which one or several amino acids are lost, substituted or added and having neurocyte function-activating activity and is useful as a therapeutic medicine, etc., for human neurodegeneration diseases such as Alzheimer's disease and Parkinson's disease. The protein is obtained by preparing cDNA library by using mRNA of human cerebrocortex as a template, screening a fragment comprising a region of a part of 3' end of the mRNA as a probe, integrating the resultant HUCPE-1 gene into an expression vector to afford a recombinant vector and expressing the vector in a host cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a protein having a stimulating activity on nerve cell function, and a protein encoding the protein.
NA and recombinant DN for expressing the gene
It is related to A.

[0002]

2. Description of the Related Art It is said that many neurodegenerative diseases cause nerve cells to die and develop due to abnormalities in nerve cells or signal transmission between nerve cells.

[0003] As a typical example of such a neurodegenerative disease,
Examples include Parkinson's disease and Alzheimer's disease. It is said that Parkinson's disease is caused by degeneration of the substantia nigra neurons to stop producing dopamine, one of the neurotransmitters, and death of dopaminergic neurons. On the other hand, Alzheimer's disease is said to exhibit dementia symptoms mainly due to death of nerve cells in the cerebral cortex and hippocampus.

[0004] For the diseases described above, there is no effective remedy because the cause is not clear, and at present, only symptomatic treatment can be carried out. At present, nerve cell death accompanying these diseases is present. Much research has been done to determine the cause of this, but it has not yet been elucidated.

[0005]

The elucidation of the mechanism of nerve cell death which is considered to be the cause of the above-mentioned diseases, and the identification of proteins involved in the mechanism and genes encoding the same are caused by nerve cell death. This is crucial in searching for a fundamental cure for the disease. For example,
Of course, there is a possibility that the protein involved in nerve cell death may be an effective drug itself, but such a protein is
It is extremely useful when developing a substance having a function similar to that of the protein, a substance having an action of inhibiting or promoting the function, or the like as a medicine. From the above viewpoints, elucidation of proteins involved in nerve cell death and their functions is desired.

[0006]

Means for Solving the Problems The present inventors aimed at identifying proteins involved in the survival of nerve cells or maintaining the function of nerve cells, and coded for genes specifically expressed in human brain tissue. As a result of intensive studies to determine the proteins involved in maintaining the desired neuronal function among the proteins being used, the existence of a novel protein (hereinafter, referred to as HUCEP-14) and the gene encoding it (hereinafter, referred to as HUCEP-14) huce
p-14) was successfully isolated. And this HU
The present inventors have found that CEP-14 has a nerve cell function activating activity and is involved in a neurodegenerative disease, and completed the present invention.

[0007] That is, the present invention relates to (a) the novel protein HUCEP-14 having the activity of activating neuronal function, comprising the amino acid sequence of SEQ ID NO: 1, or (b) the amino acid sequence of SEQ ID NO: 1. The present invention relates to a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and having a nerve cell function activating activity.

Further, the present invention relates to the above (a) or (b)
And a gene encoding the protein represented by

Further, the present invention relates to (c) a DNA comprising the nucleotide sequence of SEQ ID NO: 2; or (d) a DNA which hybridizes with the DNA of SEQ ID NO: 2 under stringent conditions and activates neuronal functions. The present invention relates to a DNA encoding an active protein.

[0010] The present invention further relates to a transformant containing a recombinant vector containing the above gene.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The gene hucep-14 can be isolated as a cDNA fragment containing the gene from a cDNA library derived from human cerebral cortex. The cDNA library used by the present inventors was prepared based on the human cerebral cortex mRNA commercially available from Clonetech, but the human cerebral cortex mRNA commercially available from Stratagene was used. In any case, cDNA can be similarly prepared.

In the above-mentioned cDNA library, as a method for identifying a cDNA which seems to have a gene specifically expressed in human brain tissue, the method of Okubo et al. (Okubo et al., Nature Gene)
t. , 2, 173 (1992)) for analyzing the frequency of appearance of gene expression. Specifically, oligo d is added to one end of a vector plasmid that has been opened with an appropriate restriction enzyme using mRNA of human cerebral cortex as a template.
After cDNA synthesis is performed using T-bonded as a primer, cleavage is carried out with restriction enzymes MboI and BamHI. Since this vector was prepared using dam methylase-positive Escherichia coli as a host, the A residue of “GATC”, which is a recognition sequence of MboI, is methylated. Therefore, MboI cuts only the newly synthesized cDNA portion. Since the vector has only one BamHI cleavage site near the end opposite to the end to which oligo dT is bound, the enzyme cuts the vector at one position and further cuts the newly synthesized cDNA portion. If a BamHI recognition sequence is present, that site is also cleaved. BamHI and MboI are composed of the sequence "GATC" and generate the same cohesive end. To cut the plasmid with both enzymes, and then to allow DNA ligase to act, the plasmid can be closed.

In this method, a cDNA library was constructed by transforming Escherichia coli using the plasmid thus prepared. Therefore, the library starts from the poly-A site at the 3 ′ end of each mRNA and its 5 ′ end.
It includes the region up to the site where the base sequence GATC first appears in the side portion. An appropriate number of recombinants are randomly selected from the cDNA library, and the cDNA in each recombinant is extracted to determine the entire base sequence. The present method is used to determine whether a cD having a specific sequence determined in this way
This is a method for discriminating an organ-specific gene and a highly expressed gene based on how many NA fragments are identified from randomly selected recombinants. In this method, extraction of the recombinant cDNA and determination of the nucleotide sequence of the cDNA are performed as follows.
All of them are various operation methods known to those skilled in the art (Mo
circular Cloning, 2nd. ed. ,
Cold Spring Harbor Lab. Pr
ess, 1989, and other methods described in technical manuals and the like, which introduce methods standard to those skilled in the art (hereinafter, referred to as ordinary methods).

In the method of identifying a high-expressing gene, the total number of recombinants selected at random is appropriately several hundred to about 1,000, but if necessary, more recombinants may be processed. . We performed the above method and found that 77
The nucleotide sequences of all the cDNA fragments in the 0 recombinants were determined, and from those, the cDNA fragments whose frequency of appearance as cDNA having the same sequence was 2/770 or more were determined.
DNA fragments having genes specifically expressed in the human brain were selected as candidates.

[0015] As described above, the cDNA fragment
It contains only a part of the 3 'end of NA. Therefore, the present inventors obtained a full-length cDNA based on the nucleotide sequence information of the region (hereinafter, 3 ′ fragment).

This is obtained by using the above 3 ′ fragment as a probe and using the human cerebral cortex cDN available from Clonetech.
A library by plaque hybridization
The screening was performed according to a conventional method.
As a result, a DNA fragment of about 2.0 kb could be obtained. At this time, a human cerebral cortex cDNA library commercially available from Stratagene can also be used as the library.

The clone obtained by the above method was propagated by using Escherichia coli as a host, and lambda phage particles were prepared according to a conventional method. DNA is extracted from the phage particles, cut with a restriction enzyme EcoRI, and a vector commercially available from Stratagene, pBlue
Script SK was similarly incorporated into the one cut with EcoRI, and the entire nucleotide sequence was determined. All of these operations were in accordance with a conventional method.

To confirm that the gene considered to be present in the cDNA fragment selected by the above method is specifically expressed in brain tissue, the organ-specific expression frequency of the cDNA sequence was confirmed by Northern hybridization. It can be done by doing. Specifically, cDNA fragments, which are commercially available from Clonetech or Stratagene, are extracted from human organs, fractionated by agarose gel electrophoresis, transferred to a membrane filter, and then selected by the above method. Using as a probe, hybridization was carried out according to a conventional method. We used this method to examine organ specificity for expression of the cDNA sequence. As a result, it was confirmed that although the cDNA sequence was somewhat expressed in organs, organs, cells, etc. other than the brain, it was specifically expressed in the cerebral cortex.

This strongly suggests that the cDNA sequence contains a desired gene that is specifically expressed in the human brain and essential for maintaining normal brain function.

A region (O) encoding a protein in the base sequence
RF, open reading frame) can be confirmed by a general-purpose method of analyzing a nucleotide sequence using a computer program. The present inventors convinced that the gene of interest was present in the cDNA sequence, found an ORF in the sequence using a computer, and identified this gene as the gene husep-14 (human cereb).
ral protein), and the protein encoded by the gene was designated as HUCEP-14.

The gene hucep-14 is represented by SEQ ID NO: 2.
Is a gene consisting of 801 base pairs (bp). Using this gene hucep-14, by a general gene recombination technique using an appropriate host vector system,
Recombinant genes can be prepared. Suitable vectors include plasmids derived from E. coli (eg, pBR32
2, pUC118, etc.), Bacillus subtilis-derived plasmids (eg, pUB110, pC194, etc.), yeast-derived plasmids (eg, pSH19, etc.), and animal viruses such as bacteriophages, retroviruses, and vaccinia viruses. Upon recombination, a translation initiation codon and a translation termination codon can be added using an appropriate synthetic DNA adapter. In order to further express the gene, an appropriate expression promoter is connected upstream of the gene. The promoter to be used may be appropriately selected according to the host. For example, when the host is Escherichia coli, a T7 promoter, a lac promoter, a trp promoter, a λPL promoter, etc.
When the host is a bacterium belonging to the genus Bacillus, an SPO-based promoter or the like is used. When the host is a yeast, a PHO5 promoter, a GAP promoter, an ADH promoter, or the like is used. When the host is an animal cell, an SV40-derived promoter or a retrovirus is used. A promoter or the like can be used respectively.

The gene can also be expressed as a fusion protein with another protein (eg, glutathione S-transferase, protein A, etc.). The fused HUCEP-14 thus expressed can be excised using an appropriate protease (eg, thrombin, enterokinase, etc.).

A host that can be used for the expression of HUCEP-14 is Escheri , a bacterium belonging to the genus Escherichia.
Various strains of chia coli, a bacteria of the genus Bacillus B
Saccharomyces cerevisi , various strains of A. subtilis , and yeasts.
ae various strains, animal cells such as COS-7 cells, C
HO cells, PC12 cells and the like can be used.

As a method for transforming a host cell using the above-mentioned recombinant vector, a conventional method or a transformation method generally used for each host cell can be applied.

The present inventors used the gene hucep with pTrcHis (Invitrogen) as an expression vector.
-14 was recombined into a HUCEP-14 expression vector, p
Truchep14 was prepared.

The present inventors further used the gene huce using pREP10 (manufactured by Invitrogen) as an expression vector.
pREhucep14, a vector for recombining p-14 and expressing HUCEP-14 in cultured animal cells
Was prepared. Using pREhucep14, PC12, which is a neuronal cell model, was transformed using Gibco's LIPOFECTAMINE reagent.
PC12 / pREhucep14 was prepared. The transformed cells are provided with or without a selectable marker present in the vector used, or an appropriate selectable marker, and identified based on the presence or absence of these selectable markers.
Can be isolated. When the PC12 cells were transformed with pREhucep14 by the present inventors, transformants can be identified and isolated using the resistance to the antibiotic hygromycin B as an index.

The expression of the target gene in the transformed cells obtained as a result of the above operation can be confirmed by Northern hybridization, as described later in Examples. When PC12 cells used as a host and PC12 cells into which the vector pREP10 was introduced were transferred to a medium without serum from a normal growth medium, cell death occurred, but PRE transformed with pREhucep14 caused cell death.
C12 can also grow in serum-free media, for example M
TT (3- (4,5-Dimethylethyltaz
ol-2-yl) -2,5-diphenyl-tet
razoliumbromide method (Mossma)
n, T. , J. et al. Immunol Methods 65,
55-59 (1985)).

The novel protein HUCEP-14 is a protein having a molecular weight of 30254 daltons consisting of 267 amino acid residues as shown in SEQ ID NO: 1.
As described above, since neuronal PC12 transformed with the recombinant vector containing the gene hucep-14 showed significantly higher survival in the absence of serum, HUCEP-14 activated neuronal cells. It was confirmed to be a bioactive protein having activity.

In the present invention, in addition to the DNA sequence shown in SEQ ID NO: 2, a DNA that hybridizes with the DNA and encodes a physiologically active protein having a nerve cell function activating activity is also included in the present invention. Within range.

That is, in the full-length sequence of the gene hucep-14, various artificial treatments such as site-directed mutagenesis, random mutation by treatment with a mutagen, mutation / deletion / ligation of a DNA fragment by restriction enzyme cleavage, etc. Even if the DNA sequence is partially changed,
If the mutant is a DNA that hybridizes with the gene hucep-14 under stringent conditions and encodes a bioactive protein having a nerve cell function activating activity, regardless of the difference from the DNA sequence shown in Sequence Listing 2, Without
It is within the scope of the present invention.

A gene consisting of a sequence slightly different from the DNA sequence shown in SEQ ID NO: 2 may exist on the human chromosome separately from the gene hucep-14. If the protein encoded therein is a physiologically active protein having a nerve cell function activating activity, it is within the scope of the present invention, similarly to the above-mentioned artificial mutant.

The degree of the above DNA mutation is determined by the gene huc
If it has a homology of 90% or more with the DNA sequence of ep-14, it is within the allowable range. Also, the gene huce
The degree of hybridization with p-14 can be determined under normal conditions (for example, DIG DNA Labeling k).
it (Cat No. manufactured by Boehringer Mannheim)
When the probe is labeled in (117533), 32
C. in a DIG EasyHyb solution (Cat No. 1603558 manufactured by Boehringer Mannheim) at 50 ° C., and a 0.5 × SSC solution (0.1
% [W / v] SDS) (with 1 × SSC 0.15 M NaCl, 0.015 M)
M is sodium sodium citrate) as long as it hybridizes to the gene hucep-14.

Also, as described above, the gene hucep-1
A physiologically active protein encoded by a mutant gene having a high homology to No. 4 and having a nerve cell function activating activity is also within the scope of the present invention.

That is, even if a mutant in which one or more amino acids of the amino acid sequence of the novel protein HUCEP-14 is deleted, substituted or added, the mutant is a protein having a nerve cell function activating activity. If so, the variant is within the scope of the invention.

The side chains of amino acids that are constituents of a protein are different from each other in hydrophobicity, charge, size, etc., but substantially affect the three-dimensional structure (also referred to as a three-dimensional structure) of the whole protein. Some conservative relationships in the sense that they are not are known empirically and by physicochemical measurements. For example, for substitution of amino acid residues, glycine (Gly), proline (Pro), Gl
y and alanine (Ala) or valine (Val), leucine (Leu) and isoleucine (Ile), glutamic acid (Glu) and glutamine (Gln), aspartic acid (Asp) and asparagine (Asn), cysteine (C
ys) and threonine (Thr), Thr and serine (Se)
r) or Ala, lysine (Lys) and arginine (A
rg), and the like.

Therefore, even if the mutant protein is a mutant protein resulting from substitution, insertion, deletion or the like in the amino acid sequence of the novel protein HUCEP-14 shown in SEQ ID NO: 1, the mutation is HUCEP-14.
If the mutated protein is a highly conservative mutation in the three-dimensional structure of the -14 protein and the mutated protein is a bioactive protein having a nerve cell function activating activity similarly to HUCEP-14, these are within the scope of the present invention. You can say something. The degree of mutation is within a range in which homology to the amino acid sequence shown in SEQ ID NO: 1 is 90% or more.

[0037]

EFFECT OF THE INVENTION Since HUCEP-14 has a nerve cell activating activity, abnormal expression of the gene hucep-14 or dysfunction of HUCEP-14 is critical for maintaining higher brain functions. It is presumed to be a major obstacle.

Therefore, HUCEP-14 itself is considered to be useful as a therapeutic drug for neurodegenerative diseases such as ischemic brain disease, Alzheimer's disease and Parkinson's disease. In addition, it can be used to create a substance having a function similar to that of the protein, a substance that enhances the function, or a substance that promotes the expression of the gene.

Hereinafter, the present invention will be described in detail with reference to Examples, but it goes without saying that the present invention is not limited to these Examples.

[0040]

EXAMPLES Example 1 Cloning of gene husep-14 1) Determination of partial sequence of gene essential for maintaining normal function of cerebrum Using mRNA of human cerebral cortex (Clontech) as a template, the method of Okubo et al. et al.
(Ture Genet., 1992, 2, p173), a cerebral cortex cDNA library was prepared.

Next, 7
Seventy recombinants were selected and used in a conventional method (Molecular
Cloning, 2nd. Ed. , Cold Sp
ring Harbor Lab. Press, 198
9, the same applies hereinafter) to extract the recombinant DNA,
The base sequence on the 3 'side of the NA portion was determined. For sequencing, a DNA sequencer (ABI PRISM377) manufactured by PE Applied Biosystems and a reaction kit manufactured by the company were used. As a result of analyzing the expression frequency of each DNA fragment in the 770 recombinants, the sequence shown in FIG.
Was 2/770.

2) Cloning of cDNA Fragment Containing Sequence-1 The cDNA fragment containing Sequence-1 was cloned by the following method.

First, an oligonucleotide consisting of a part of sequence-1 (FIG. 1; sequence-2) was converted to a DNA synthesizer (ABI 380B) manufactured by PE Applied Biosystems.
Was synthesized. Human Braincerebral cor using λgt11 as a cloning vector
tex 5'-STRETCH cDNA library
ry (manufactured by Clontech Laboratories) with E. coli K
Plaques were formed using 12 strains, Y1090, as a host according to a conventional method. The plaque was transferred to a membrane filter (Hybond-N + manufactured by Amersham), and DIG
Using the oligonucleotide of sequence-2 labeled with (digoxigenin) as a probe, a phage having sequence-2 was obtained by plaque hybridization.
For labeling, a DIG oligonucleotide tailing kit (manufactured by Boehringer Mannheim) was used, and the method followed the procedure of this kit. Hybridization was performed at 51 ° C. for 5 hours in a solution having the following composition (all concentrations were final concentrations).

5 × SSC 1% Blocking Buffer 0.1% N-lauroylsarcosyl sodium 0.02% SDS 50 μg / ml polyA 1 pmol / ml DIG-labeled synthetic DNA After completion of hybridization, the membrane was washed with 2 × SS
C, 0.1% SDS, then 0.5 × SSC, 0.1%
Washing was performed at 51 ° C. using SDS. After washing the membrane,
DIG emission detection kit (Boehringer Mannheim)
The membrane was processed according to the procedure of the kit. For signal detection, Hyperfilm ^™ -E
CL (manufactured by Amersham) film was used.

The plaque hybridized with the probe was purified according to a conventional method to obtain a single clone.

The single clone was propagated, and DNA was extracted and purified from phage particles. All of these operations were in accordance with a conventional method.

3) Subcloning into a nucleotide sequence determination vector The DNA purified in 2) was digested with a restriction enzyme, EcoRI, and similarly digested with EcoRI, pBlue
It was subcloned into scriptSK (Stratagene). The ligation solution is a kit (Takara DNA Ligation Kit) manufactured by Takara Shuzo Co., Ltd.
Ver. Using 2), the reaction was carried out at 16 ° C. for 1.5 hours.

Using the above reaction solution, Escherichia coli K12 strain DH5 was transformed according to a conventional method.

The transformant was treated with ampicillin (Amp) 50
μg / ml, 5-Bromo-4-Chloro-3-
indolyl-β-D-galactoside (X
-Gal) 40 μg / ml, Isopropyl-β-
D-Thio-Galactopyranoside
(IPTG) The cells were plated on LB agar medium containing 100 μM, and cultured at 37 ° C. overnight.

The white colonies were inoculated into 10 ml of an LB liquid medium containing 50 μg / ml of Amp, cultured at 37 ° C. overnight, and the cells were collected by centrifugation, followed by QIAprep.
Spin Plasmid Miniprep Ki
The recombinant DNA was purified by t (manufactured by Qiagen). The thus-constructed recombinant DNA was ligated with pBShucep14.
(FIG. 3).

4) Determination of Base Sequence of DNA Fragment The base sequence was determined using a DNA sequencer manufactured by PE Applied Biosystems, using the dye terminator method. Oligonucleotides were synthesized based on the determined base sequence, and the entire base sequence was determined by the primer walking method (FIG. 4). The nucleotide sequences of both strands were determined. FIG. 5 shows the entire nucleotide sequence of the cDNA of the clone. Since the base sequence contained sequence-2, the target gene (human cerebral protein) was used.
in-14, hucep-14) was confirmed to be cloned.

The cDNA has a translation region (open) encoding a protein (HUCEP-14) consisting of 267 residues.
reading frame (ORF) (FIG. 5).

5) Production of HUCEP-14 Using Escherichia coli Based on the sequence shown in FIG. 5, oligonucleotides of sequence-3 and sequence-4 were converted to a DNA synthesizer (ABI 380B, manufactured by PE Applied Biosystems). Was synthesized.

Sequence-3 5'CCGGCGCAGCCATGGTGAAGATTAGC Sequence-4 5'CTCACACCACCCCGCAGATGAGCGTC Recombinant DNA prepared in Example 1-2), pBshuc
PCR was carried out under the following reaction conditions using ep14 as a template and oligonucleotides of sequence-3 and sequence-4 as primers. The reaction was carried out using Stratagene's Pfu D
A PCR thermal cycler MP manufactured by Takara Shuzo Co., Ltd. was used using NA polymerase.

Reaction composition pBShucep14 (1 μg / ml) 1 μl 10 × PCR buffer 5 μl 2.5 mM dNTP 8 μl 10 μM Conditions After holding at 94 ° C. for 1 minute, the sample was cooled to 53 ° C. at a rate of −1 ° C./2 seconds, kept at 53 ° C. for 1 minute, and further kept at 72 ° C. for 3 minutes. After repeating this 30 times, the target sequence was amplified by holding at 72 ° C. for 10 minutes.

The DNA fragment amplified by the above method was fractionated by agarose gel electrophoresis to obtain a DNA fragment of about 0.8 kb.
The fragment was purified (fragment-1). Fragment-1 was mixed with the previously opened vector, pCR-Blunt (manufactured by Invitrogen), and ligated and transformed into Escherichia coli K12 strain DH5 under the conditions described in Example 1-3). The transformant was cultured and the recombinant DNA was purified from the cells collected by centrifugation. Using the base sequence of fragment-1 inserted into the recombinant DNA, primers of sequence-5 and sequence-6 shown below, and primers used in determining the base sequence in Example 1-4) And determined with a DNA sequencer.

Sequence-5 5'CACAGGAAACAGCTATGACC Sequence-6 5'ATACGACTCACTATAGGGCG As a result, the nucleotide sequence of the fragment-1 was hucep-14.
Was confirmed to be the same as the base sequence. Of the recombinant DNA thus constructed, fragment-1 was inserted such that the start codon of HUCEP-14 was located on the BamHI cleavage site side of pCR-Blunt, and the stop codon was located on the XhoI cleavage site side. Recombinant DNA was pCRhu
It was named cep14 (FIG. 6). The cells carrying the recombinant DNA were cultured, and the recombinant DNA was purified from the cells collected by centrifugation and cut with restriction enzymes BamHI and XhoI (both from Takara Shuzo). After the cleavage treatment, fractionation was performed by agarose gel electrophoresis, and a DNA fragment of about 0.8 kb was purified (fragment-2). PTrcHi, a vector for protein production
sA (manufactured by Invitrogen) was digested with restriction enzymes BamHI and XhoI in the same manner as described above, and the ring-opened vector (fragment-3) was purified. Fragment-2 and fragment-3 were mixed, ligation and transformation of E. coli K12 strain DH5 were performed under the conditions described in Example 1-3), and the transformants were further cultured and collected by centrifugation. The recombinant DNA was purified from the cells. The recombinant DNA thus constructed was named pTrchucep14 (FIG. 7).
The cells holding the recombinant DNA are cultured, and IPTG
Was added to the culture solution to a final concentration of 0.1 to 1 mM to induce gene expression.
No. 4 can be produced as a fusion protein in which an oligopeptide having an enterokinase cleavage site on the N-terminal side and 6 histidine residues further upstream thereof are bound.

Example 2 hucep in PC12 cells
Analysis of Expression and Function of -14 Gene 1) Construction of Expression Vector pREhucep14 Expression vector pREP10 (manufactured by Invitrogen) of animal cells is cut with restriction enzymes BamHI and XhoI (both manufactured by Takara Shuzo), and a ring-opening vector (fragment- 4) was purified.

Fragment-2 and fragment-4 purified in Example 1 were mixed, ligated and transformed into Escherichia coli K12 strain DH5 under the conditions described in Example 1, and the transformant was further cultured. The recombinant DNA was purified from the cells collected by centrifugation. The recombinant DNA thus constructed was named pREhucep14 (FIG. 8).

2) Introduction into PC12 Cells and Obtaining Stable Transformants PC12 cells were cultured in a plastic Petri dish having a diameter of 60 mm. The dishes were collagen-coated, and the medium used was 5% fetal calf serum, 5% horse serum, 50%
The cells were cultured at 37 ° C. in the presence of 5% CO ₂ using DMEM (manufactured by Gibco, hereinafter referred to as a growth medium) containing unit / ml penicillin and 50 μg / ml streptomycin.

When the cell density reaches 50%, LIPOFECT containing pREhucep14 constructed in 1)
AMINE reagent (manufactured by Gibco) is layered on the cells and
After culturing for 4 hours, the medium was replaced with a growth medium and cultured for 24 hours. After dispersing the cells by pipetting, the cell suspension was divided into two equal parts, dispensed into two plastic dishes having a diameter of 100 mm, and further cultured for 24 hours.

After removing the medium, the medium was replaced with a growth medium containing hygromycin B (manufactured by Calbiochem; final concentration: 400 μg / ml). Add 3 media of Hygromycin B
The culture was changed every day and cultured for 2 weeks. When the cell colonies became visible with the naked eye, five colonies were isolated using a stainless steel cup. For use as a control, pREP10 alone was introduced into PC12 cells in the same manner as described above, and five stable transformants were isolated.

3) Confirmation of gene expression Each isolated transformant was cultured in a 24-well plate in a medium containing hygromycin B (final concentration: 400 μg / ml). When the cell density reached 80% confluence, The cells were dispersed by pipetting and inoculated into a plastic petri dish having a diameter of 100 mm. When the cell density became 80% confluent again, the medium was removed, PBS was added, and the cells were collected using a cell scraper. The supernatant is removed after sedimenting the cells by centrifugation and the mRNA is removed.
MRNA was purified from the cells using an extraction kit (Pharmacia Biotech). 2 μg of mRNA was fractionated by agarose gel electrophoresis according to a standard method, transferred to a membrane (Hybond-N + manufactured by Amersham), and subjected to Northern hybridization. As a probe, hucep-1 labeled with DIG (digoxigenin) was used.
4 cDNA fragments were used. For labeling, a DIG oligonucleotide tailing kit (manufactured by Boehringer Mannheim) was used, and the method followed the procedure of this kit. Hybridization was performed at 51 ° C. for 5 hours in a solution having the following composition (all concentrations were final concentrations).

5 × SSC 1% Blocking Buffer 0.1% N-lauroylsarcosyl sodium 0.02% SDS 50 μg / ml polyA 1 pmol / ml DIG-labeled synthetic DNA After completion of hybridization, the membrane was washed with 2 × SS
C, 0.1% SDS, then 0.5 × SSC, 0.1%
Washing was performed at 51 ° C. using SDS.

After washing the membrane, the membrane was treated using a DIG emission detection kit (manufactured by Boehringer Mannheim) according to the procedure of the kit. Hyperfilm ^™ -ECL (Amersham) film was used for signal detection.

As a result, the PC12 cells into which pREhucep14 had been introduced were better than the PC1 cells into which pREP10 had been introduced.
The expression level of the hucep-14 gene was higher than in the two cells.

[0067]

[Sequence list]

 SEQUENCE LISTING <110> Taisho Phermaceutical Co., LTD <120> Neuroprotective Protein <130> 00TS-P2744 <140> JP <141> 1998-9-8 <150> JP <151> 1997-9-8 <160> 2 <210> 1 <211> 267 <212> PRT <213> HUMAN <400> Met Val Lys Ile Ser Phe Gln Pro Ala Val Ala Gly Ile Lys Gly Asp 1 5 10 15 Lys Ala Asp Lys Ala Ser Ala Ser Ala Pro Ala Pro Ala Ser Ala Thr 20 25 30 Glu Ile Leu Leu Thr Pro Ala Arg Glu Glu Gln Pro Pro Gln His Arg 35 40 45 Ser Lys Arg Gly Ser Ser Val Gly Gly Val Cys Tyr Leu Ser Met Gly 50 55 60 Met Val Val Leu Leu Met Gly Leu Val Phe Ala Ser Val Tyr Ile Tyr 65 70 75 80 Arg Tyr Phe Phe Leu Ala Gln Leu Ala Arg Asp Asn Phe Phe Arg Cys 85 90 95 Gly Val Leu Tyr Glu Asp Ser Leu Ser Ser Gln Val Arg Thr Gln Met 100 105 110 Glu Leu Glu Glu Asp Val Lys Ile Tyr Leu Asp Glu Asn Tyr Glu Arg 115 120 125 Ile Asn Val Pro Val Pro Gln Phe Gly Gly Gly Asp Pro Ala Asp Ile 130 135 140 Ile His Asp Phe Gln Arg Gly Leu Thr Ala Tyr His Asp Ile Ser Leu 145 150 155 160 Asp Lys Cys Tyr Val Ile Glu Leu Asn Thr Thr Ile Val Leu Pro Pro 165 170 175 Arg Asn Phe Trp Glu Leu Leu Met Asn Val Lys Arg Gly Thr Tyr Leu 180 185 190 Pro Gln Thr Tyr Ile Ile Gln Glu Glu Met Val Val Thr Glu His Val 195 200 205 Ser Asp Lys Glu Ala Leu Gly Ser Phe Ile Tyr His Leu Cys Asn Gly 210 215 220 Lys Asp Thr Tyr Arg Leu Arg Arg Arg Ala Thr Arg Arg Arg Ile Asn 225 230 235 240 Lys Arg Gly Ala Lys Asn Cys Asn Ala Ile Arg His Phe Glu Asn Thr 245 250 255 Phe Val Val Glu Thr Leu Ile Cys Gly Val Val 260 265 <210> 2 <211> 801 <212> DNA <213> HUMAN <400> atggtgaaga ttagcttcca gcccgccgtg gctggcatca agggcgacaa 50 ggctgacaag gcgtcgg gccgcccgcccgcccgcccgcccgcccgccc agggggagct cagtgggcgg cgtgtgctac ctgtcgatgg gcatggtcgt 200 gctgctcatg ggcctcgtgt tcgcctctgt ctacatctac agatacttct 250 ttcttgcaca gctggcccga gataacttct tccgctgtgg tgtgctgtat 300 gaggactccc tgtcctccca ggtccggact cagatggagc tggaagagga 350 tgtgaaaatc tacctcgacg agaactacga gcgcatcaac gtgcctgtgc 400 cccagtttgg cggcggtgac cctgcagaca t catccatga cttccagcgg 450 ggtctgactg cgtaccatga tatctccctg gacaagtgct atgtcatcga 500 actcaacacc accattgtgc tgccccctcg caacttctgg gagctcctca 550 tgaacgtgaa gagggggacc tacctgccgc agacgtacat catccaggag 600 gagatggtgg tcacggagca tgtcagtgac aaggaggccc tggggtcctt 650 catctaccac ctgtgcaacg ggaaagacac ctaccggctc cggcgccggg 700 caacgcggag gcggatcaac aagcgtgggg ccaagaactg caatgccatc 750 cgccacttcg agaacacctt cgtggtggag acgctcatct gcggggtggt 800 g 801

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a DNA fragment which shows a high expression frequency in a recombinant obtained from a cerebral cortex cDNA library.
The oligonucleotide used for the cloning of the A fragment is shown.

FIG. 2 shows a DNA fragment containing Sequence-1.

FIG. 3 is a continuation of FIG. 2;

FIG. 4 shows the construction of recombinant DNA, pBShucep14.

FIG. 5 shows a method for determining the nucleotide sequence of gene hucep-14.

FIG. 6 shows the nucleotide sequence of the gene hucep-14 and the amino acid sequence encoded thereby.

FIG. 7 is a continuation of FIG. 5 (1).

FIG. 8 is a continuation of FIG. 5 (2).

FIG. 9 is a continuation of FIG. 5 (3).

FIG. 10 shows the construction of the recombinant DNA, pCRhucep14.

FIG. 11 shows the construction of a recombinant DNA, pTrchucep14.

FIG. 12 shows the construction of a recombinant DNA, pREhucep14.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // A61K 38/22 AAM A61K 37/24 AAM (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19) (C12P 21/02 C12R 1:91) (72) Inventor Yoshiyoshi Takayama 3- 24-1, Takada, Toshima-ku, Tokyo Inside Taisho Pharmaceutical Co., Ltd. (72) Inventor Katsuki Tsuritani 3--24 Takada, Toshima-ku, Tokyo No. 1 Taisho Pharmaceutical Co., Ltd.

Claims (4)

[Claims] 1. A protein of the following (a) or (b): (a) a protein comprising the amino acid sequence of SEQ ID NO: 1; (b) one or several amino acids in the amino acid sequence of SEQ ID NO: 1 A protein comprising an amino acid sequence in which is deleted, substituted or added, and having a nerve cell function activating activity. 2. A gene encoding the protein of (a) or (b) according to claim 1. 3. A gene consisting of the following (a) or (b): (a) a DNA consisting of the nucleotide sequence of SEQ ID NO: 2; (b) hybridizing with the DNA of SEQ ID NO: 2 under stringent conditions And a human-derived DNA encoding a protein having a nerve cell function activating activity. 4. A transformant comprising a recombinant vector containing the gene according to claim 2 or 3.