JP3131770B2 - Protein with neurite outgrowth activity - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a protein having a neurite outgrowth activity, a DNA encoding the protein, a neurite outgrowth agent containing the protein as an active ingredient, an anti-dementia agent, a memory improving agent and a brain function activator. About.

[0002]

2. Description of the Related Art In the course of development, nerve cells are extended by neurites under the influence of the external environment and form neural circuits. In addition, it is considered that morphological changes of synapses, in particular, elongation of new nerve endings called germination and formation of synapses to new places are important in information processing. It is thought that synapse formation associated with neurite outgrowth is closely related to memory, and since neurites are lost in patients with dementia, it is possible to improve memory or treat dementia by forming neurites. It is possible to do. The role of a protein having neurite outgrowth activity is considered important for neurite outgrowth, but its substance has not been elucidated.

The present inventor reported a protein having a neurite outgrowth activity (Japanese Patent Application Laid-Open No. 8-127595), and by further research, newly found a protein having a completely new sequence.

An object of the present invention is to provide a protein having neurite outgrowth activity and a DNA encoding the protein.

Another object of the present invention is to provide a neurite elongation agent, an anti-dementia agent, a memory improving agent and a brain function activating agent.

[0006]

Means for Solving the Problems As a result of repeated studies in view of the above problems, the present inventor has found that DN encoding a novel protein
A. It was found that a protein isolated from the DNA sequence significantly promoted neurite outgrowth.

That is, the present invention provides the following items 1 to 10
A protein, DNA, neurite outgrowth agent, anti-dementia agent, memory improving agent and brain function activator. .

Item 1. A protein having neurite outgrowth activity, represented by any of SEQ ID NOs: 1 to 676, or a protein derivative in which one or several amino acids have been substituted, deleted, or added.

Item 2. A protein having neurite outgrowth activity, represented by 249 to 676 of SEQ ID NO: 1, or
The protein derivative in which one or several amino acids have been substituted, deleted or added.

Item 3. A DNA having a neurite outgrowth activity, comprising a nucleic acid sequence encoding a protein represented by SEQ ID NO: 1 to 676 or a protein derivative in which one or several amino acids have been substituted, deleted or added.

Item 4. Proteins having neurite outgrowth activity represented by 249 to 676 of SEQ ID NO: 1 or 1
Alternatively, a DNA comprising a nucleic acid sequence encoding the protein derivative in which several amino acids have been substituted, deleted or added.

Item 5. Item 6. A DN capable of hybridizing with the DNA according to item 3 or 4 under stringent conditions
A.

Item 6. Item 5. The DNA according to item 4, represented by SEQ ID NO: 1.

Item 7. A neurite outgrowth agent comprising the protein according to item 1 or 2 as an active ingredient.

Item 8. Item 13. An anti-dementia agent comprising the protein according to Item 1 or 2 as an active ingredient.

Item 9. Item 3. A memory improving agent comprising the protein according to Item 1 or 2 as an active ingredient.

Item 10. Item 3. A brain function activator comprising the protein according to Item 1 or 2 as an active ingredient.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION Regarding the neurite elongation activity of the protein of the present invention, the C-terminal 249 to 676 shown in SEQ ID NO: 1
With the amino acid sequence of neurite outgrowth activity,
1-676 proteins have even more potent neurite outgrowth activity.

[0019] The protein of the present invention comprises 249 to
676 or 1 to 676 at one or several positions at a specific site or at random substitution, addition or deletion, as long as it has neurite outgrowth activity,
It is included in the scope of the present invention.

As a method for substituting, adding or deleting a specific amino acid, conventionally known methods such as a point mutation method and a deletion / insertion method using PCR are used.

In the present specification, "stringent conditions" means conditions usually used in a hybridization method, and such conditions can be easily understood by those skilled in the art.

The protein of the present invention can be obtained from a chick denervated muscle extract using, for example, a primary culture system of chicken embryo telencephalic neurons as an assay system, but is not limited to chick-derived proteins. . The sequence of the protein having an activity to form neurites has been revealed for the first time by the present inventors, and is derived from all animals such as birds such as chickens, mammals such as humans, mice, rats, rabbits and hamsters. Any protein having a projection elongation activity and having homology to the protein is included in the present invention.

The protein of the present invention can be obtained, for example, by introducing a vector into which a DNA encoding the protein having neurite outgrowth activity has been incorporated into cells to obtain a transformant, and culturing the transformant in a medium. It is produced by the method for producing the protein represented by the above-mentioned item 1 as a feature.

The present inventors isolated a monoclonal antibody against a protein having neurite outgrowth activity using chick denervated muscle extract, and used the monoclonal antibody to isolate c
The DNA library was screened and c shown in FIG.
DNA is obtained and 249-676 encoded by the cDNA
And proteins having the sequence of 1-676 were isolated and confirmed to have neurite outgrowth activity.

The cell transformed with the vector incorporating the DNA encoding the protein having neurite elongation activity is not particularly limited, and conventionally known cells for transformation are widely used. Examples include bacteria such as Escherichia coli, eukaryotic microorganisms such as yeast, and cultured cells of various mammals such as mice, rats, hamsters, and humans.

The introduction of the vector into Escherichia coli or the like can be carried out according to a known method.

The vector used for production of the protein of the present invention is not particularly limited as long as it has a promoter necessary for translation of the DNA encoding the protein of the present invention. Examples thereof include pBluescript and pGEX. .

[0028] The present invention also relates to a transformant in which a vector incorporating a DNA encoding the protein having neurite outgrowth activity has been integrated into the cell.

The medium in which the transformant is cultured depends on the type of cells to be transformed. For example, in the case of a microorganism such as Escherichia coli, a carbon source (such as glucose), a nitrogen source (such as ammonium sulfate), A medium containing an inorganic substance (sodium phosphate, iron sulfate, manganese sulfate, etc.) can be used. As the culture conditions such as temperature, pH and time, ordinary culture conditions for various cells are used as they are.

The protein of the present invention has a neurite elongation activity, and the protein can be used as a neurite elongation agent. In addition, it has been revealed that the formation of a neural circuit by neurite outgrowth is also closely related to memory and dementia, and the protein is used as an anti-dementia agent, a memory improving agent, a nerve fiber repair agent, and a nerve cell. It is also used as an activator, learning improver, emotional disorder improver, and the like.

The protein of the present invention may be used as an injection, as a neurite elongation agent, an anti-dementia agent, a memory improving agent or a brain function activator.
Oral preparations (tablets, granules, capsules, solutions, suspensions, etc.), ointments, suppositories and the like can be administered. The protein of the present invention is preferably administered as an injection. These dosage forms can be easily prepared by appropriately using a pharmaceutically acceptable carrier usually used for formulation with the protein of the present invention.

For example, when preparing an injection, a pH adjuster, a buffer, a stabilizer, an isotonic agent,
Add a local anesthetic, subcutaneous, intramuscular,
An intravenous injection can be prepared.

The protein of the present invention is administered at a concentration of about 1 to 10 μg / ml in order to exhibit the above-mentioned biological activity. When administered as an anti-dementia agent, a memory improving agent or a brain function activator, it is preferable to administer about 0.0001 mg to 10 mg per day for an adult as an injection.

[0034]

According to the present invention, a novel protein showing neurite elongation activity was isolated and its structure was clarified.

The protein can be easily mass-produced by a genetic engineering method.

The protein is useful as an anti-dementia agent, a memory improving agent, a brain function activating agent, a nerve fiber repairing agent, a nerve cell activating agent, a learning improving agent, an emotional ameliorating agent and the like.

[0037]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 (1) Preparation of Monoclonal Antibody (1-1) Preparation of Antigen A 5 day-old chick sciatic nerve innervating the leg muscles was cut, and after 3 days the leg muscles were cut off to remove protease. Was homogenized using a mixer in a HEPES buffer solution (pH 7.0) containing, and then centrifuged (100,000 g, 1 hour, 4 ° C), and the obtained supernatant was used as an antigen.

(1-2) Mouse Immunization Immunization was performed by injecting 0.2 ml of an antigen (2 mg / ml) mixed with an equal amount of Freund's adjuvant into the peritoneal cavity of a mouse (BALB / c). A small amount of blood was collected from the tail of a mouse anesthetized with ether and used to prepare serum. The increase in the antibody titer was confirmed using a usual ELISA method.

(1-3) Preparation of Hybridoma A spleen (1: 2500 in this experimental example) of a mouse whose antibody titer was raised to 1: 1000 was cut out, cut under aseptic conditions, and subjected to stainless mesh (# 100). 5ml on
Dissociated splenocytes were obtained by crushing using a syringe piston. Myeloma cells (PAI cells) started to proliferate 10 days before the preparation of spleen cells. The prepared dissociated splenocytes and myeloma cells were prepared using polyethylene glycol 1500
(Final concentration 9% (v / v)).

(1-4) Screening After fusion, the hybridomas were cultured at a density of 4 cells / well.
A part of the culture solution of the wells that had been cultured in an O ₂ incubator and in which colony formation was confirmed was collected, and the production of the antibody was confirmed by ELISA. Cells in positive wells in which antibody production was confirmed were advanced to cloning and secondary screening. The finally obtained clone was isolated after the third screening.

(1-5) Selection of Activity-Inhibiting Antibody Among the obtained monoclonal antibodies, those that inhibit neurite outgrowth activity were selected by the following method. The antigen was placed in a culture dish, a protein having neurite outgrowth activity was adhered to the bottom surface, a monoclonal antibody was added, and the mixture was allowed to stand for 1 hour. The solution in the culture dish was exchanged for a culture solution, telencephalic neurons of chicken embryos were added, and after culturing for 24 hours, the state of neurite outgrowth was observed. The 4D2a monoclonal antibody suppressed the elongation activity most strongly.

(2) cDN from cDNA library
Isolation of A (2-1) Preparation of cDNA library The sciatic nerve innervating the 5-day-old chick leg muscle was cut, and three days later, the leg muscle was removed, and after homogenization, Cs was removed.
R in the density gradient centrifugation (45,000 rpm; 40 hours) using Cl.
NA was separated. Further, mRNA was isolated using an oligo dT column according to a conventional method, cDNA was synthesized using reverse transcriptase (manufactured by Stratagene), and the obtained cDNA was incorporated into a λZAP vector. A λ phage particle was formed by in vitro packaging, and this was used to infect Escherichia coli to prepare a cDNA library.

(2-2) Screening The target clone was isolated by the plaque screening method. At this time, a 4D2a monoclonal antibody was used. Screening was performed twice, and clones that reliably expressed the protein recognized by this antibody were selected. The pBluescript portion in the phage vector was isolated by allowing helper phage (f1) to act.

(3) Introduction of a vector containing the DNA into E. coli (3-1) Subcloning into an expression vector (for expression of a protein having an amino acid sequence of 1 to 676) 196 bp to 2226 bp of the cloned cDNA
An EcoRI adapter was added to the 5 'end and a poly A tail + Xho I site was added to the 3' end.
It was subcloned into the EcoRI-XhoI site of GEX4T3.

(In the case of expressing a protein having an amino acid sequence of 249 to 676) 747 of the cloned cDNA
EcoRI adapter, 3 'at 5' end of bp-2226bp
Add a poly A tail + Xho I site to the end
Was subcloned into the EcoRI-XhoI site of the expression vector pGEX4T3.

(3-2) Expression Using each of the above plasmids, competent E. coli JM
105 was transformed, and a fusion protein of glutathione-S-transferase and the target protein was expressed with isopropyl-β-D-thiogalactoside.

(4) Isolation of target protein Escherichia coli expressing the fusion protein was disrupted by ultrasonication.
The soluble fraction was separated. This fraction was passed through a glutathione-Sepharose 4B column, and the recombinant protein was purified using the affinity of glutathione-S-transferase (GST) and glutathione.

(5) Method for measuring neurite outgrowth activity of isolated proteins Purified recombinant proteins (1-676, 249-67)
6. PD (denervated ganglion extract, GST) was diluted with Ham's F12 medium (provided that 10 mM glucose and 2.5 μg / ml insulin were added) and added to a culture dish (16 mm diameter).
The mixture was allowed to stand at 0 ° C for 2 hours. On the other hand, the cerebrum (teural brain) and spinal cord (including motor nerve) were cut out from the chicken embryo on day 5 of the egg, treated with 0.0125% trypsin at 37 ° C. for 30 minutes, washed with the above-mentioned F12 medium, and then pipetted. Dispersed. Put 4 × 10 ⁴ neurons in each well of the previous culture dish,
The cells were cultured for 24 hours under the conditions of 37 ° C., 5% CO ₂ , and 100% humidity. After the culture, the cells were observed with a phase-contrast microscope, and the proportion of nerve cells having a neurite longer than twice the cell body was measured. The results are shown in FIGS.

[0050]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 4723 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear CACAGCCAGT CCCCTGCACA GTGTGCAGTT GGGCTCTGCA GTGGTTGTTC TTGCTGTGTC 60 ACTCTCGCCT TCCCCCTTGC TTGGTCTTCT TGGTGGCAGG ATTATATCTCTGTCTC TCG TGT GTC TGC TGT GTC GTC TGC TGT GTC GTC TCG TCG TGT GTC ATG GAG AAT GAC CAG TTC ACC GAG AAG CAG CAG CAG 231 Met Glu Asn Asp Gln Phe Thr Glu Lys Gln Gln Gln 1 5 10 GTT ACC ACC TCA CCT ACG CAG GAC AAC CAG GGG CAA AGC AAG GCG GAA 279 Val Thr Thr Ser Pro Thr Gln Asp Asn Gln Gly Gln Ser Lys Ala Glu 15 20 25 CCT GTC CCC GTC TCA CAG CCC CTT TCC CCC ACA AAC CAA ACC AGT GCC 327 Pro Val Pro Val Ser Gln Pro Leu Ser Pro Thr Asn Gln Thr Ser Ala 30 35 40 CAG CCT GAG ATG GCC ACG TGT GAC ATC TCA GAG GAG CTG AAC CGG CAA 375 Gln Pro Glu Met Ala Thr Cys Asp Ile Ser Glu Glu Leu Asn Arg Gln 45 50 55 60 CTG GAG GAC ATT ATT AAA ACA TAT GGG TCT GCA GCG AGT CTG GTA GAG 423 Leu Glu Asp Ile Ile Lys Thr Tyr Gly Ser Ala Ala Ser Leu Val G lu 65 70 75 AAG GAA GGC ACT ACA GCA GAA ACT GAC AAG CCA GAG AAG GAA GAT GTG 471 Lys Glu Gly Thr Thr Ala Glu Thr Asp Lys Pro Glu Lys Glu Asp Val 80 85 90 GGC AGT ATG GAG GAT GCA GAG TGT GAG GAT GTA AAT GAA GAA AGT GAG 519 Gly Ser Met Glu Asp Ala Glu Cys Glu Asp Val Asn Glu Glu Ser Glu 95 100 105 AAA GAC AAA CCA GCT CCT GGA GAT GCT TCA AGA GCA AAG GAG CCC AGT 567 Lys Asp Lys Pro Ala Pro Gly Asp Ala Ser Arg Ala Lys Glu Pro Ser 110 115 120 GCC AGC AAG GAA CAA AAG CTG GAG AAG AAA ATC CTG AAA GGA CTA GGG 615 Ala Ser Lys Glu Gln Lys Leu Glu Lys Lys Ile Leu Lys Gly Leu Gly 125 130 135 140 AAG GAA GCC ACC CTC CTC ATG CAA AGC TTG AAC AAG CTG ACT ACT CCA 663 Lys Glu Ala Thr Leu Leu Met Gln Ser Leu Asn Lys Leu Thr Thr Pro 145 150 155 GAG GAG AAG CTG GAC CTG TTA TTT AAG AAG TAT GCT GAG TTG CTT GAG 711 Glu Glu Lys Leu Asp Leu Leu Phe Lys Lys Tyr Ala Glu Leu Leu Glu 160 165 170 GAG CAT CGT GCT GAG CAG AAG CAG CTC AAG TAC CTG CAG AAG AGG CAG 759 Glu His Arg Ala Glu Gln Lys Gln Leu Lys Tyr Le Gln Lys Arg Gln 175 180 185 GCC CAG ATC ACC AAG GAG AAG GAC CAG TTG CAG AGT GAG CAC AGC CGA 807 Ala Gln Ile Thr Lys Glu Lys Asp Gln Leu Gln Ser Glu His Ser Arg 190 195 200 GCC ATC CTT GCT CGC AGC AAG CTT GAG AGC CTG TGC CGG GAG CTC CAG 855 Ala Ile Leu Ala Arg Ser Lys Leu Glu Ser Leu Cys Arg Glu Leu Gln 205 210 215 220 AGG CAC AAC AAA ACC CTC AAG GAG GAA ACA ATT CAG CGG GCA CGG GAG 903 Arg His Asn Lys Thr Leu Lys Glu Glu Thr Ile Gln Arg Ala Arg Glu 225 230 235 GAA GAT GAG AAG AGG AAA GAA ATA ACA AAT CAT TTC CAG GGC ACG CTG 951 Glu Asp Glu Lys Arg Lys Glu Ile Thr Asn His Phe Gln Gly Thr Leu 240 245 250 AGT GAA ATC CAG GCT CAG ATT GAG CAG CAA AGT GAG AGG AAC ATG AAG 999 Ser Glu Ile Gln Ala Gln Ile Glu Gln Gln Ser Glu Arg Asn Met Lys 255 260 265 CTC TGC CAG GAG AAC ACA GAG CTG GCA GAG AAG CTG AAA GC ATT ATT 1047 Leu Cys Gln Glu Asn Thr Glu Leu Ala Glu Lys Leu Lys Ser Ile Ile 270 275 280 GAC CAG TAT GAG CTG CGG GAA GAG CAC CTT GAC AAA ATA TTT AAG CAC 1095 Asp Gln Tyr Glu Leu Arg Glu Glu His Leu As p Lys Ile Phe Lys His 285 290 295 300 300 AGA GAA CTT CAA CAG AAA TTG GTG GAT GCC AAG TTG GAG CAG TCT CAG 1143 Arg Glu Leu Gln Gln Lys Leu Val Asp Ala Lys Leu Glu Gln Ser Gln 305 310 315 GAA ATG ATG AAG GAA GCA GAG GAG CGA CAT CAG AAG GAA AAG GAA TAT 1191 Glu Met Met Lys Glu Ala Glu Glu Arg His Gln Lys Glu Lys Glu Tyr 320 325 330 CTC CTG AAT CAA GCC GCA GAA TGG AAG CTA CAG GCC AAA ATG TTA AAG 1239 Leu Leu Asn Gln Ala Ala Glu Trp Lys Leu Gln Ala Lys Met Leu Lys 335 340 345 GAA CAG GAG ACA GTC CTG CAG GCA CAG ATC ACT CTC TAC TAC TCT GAG AGA 1287 Glu Gln Glu Thr Val Leu Gln Ala Gln Ile Thr Leu Tyr Ser Glu Arg 350 355 360 TTT GAA GAA TTT CAG AAA ACA TTG ACC AAA AGC AAT GAA GTG TTT GCT 1335 Phe Glu Glu Phe Gln Lys Thr Leu Thr Lys Ser Asn Glu Val Phe Ala 365 370 375 380 ACC TTC AAA CAG GAG ATG GAG AAA ATG ACA AAG AAA ATG AAG AAG TTG 1383 Thr Phe Lys Gln Glu Met Glu Lys Met Thr Lys Lys Met Lys Lys Leu 385 390 395 GAA AAG GAT ACT GCT ACA TGG AAA TCC AGG TTT GAG AAC TGT AAC AGA 1431 Glu Lys Asp Thr Al a Thr Trp Lys Ser Arg Phe Glu Asn Cys Asn Arg 400 405 410 GCA TTA CTG GAC ATG ATT GAA GAG AAA GCC ATG AGG ACC AAG GAA TAC 1479 Ala Leu Leu Asp Met Ile Glu Glu Lys Ala Met Arg Thr Lys Glu Tyr 415 420 425 GAG TGC TTT GTG CTG AAA ATC CAG AGG CTA GAG AAC CTT TGC CGA GCT 1527 Glu Cys Phe Val Leu Lys Ile Gln Arg Leu Glu Asn Leu Cys Arg Ala 430 435 440 CTG CAG GAA GAG AGG AAT GAA TTG TAC AGA AAA ATA AAA CAA GCA CAG 1575 Leu Gln Glu Glu Arg Asn Glu Leu Tyr Arg Lys Ile Lys Gln Ala Gln 445 450 455 460 CTC CCT GAA GAG GTG AAT GGA AAT GAC ATC TTA GAA GAA GAC GAC GAT 1623 Leu Pro Glu Glu Val Asn Gly Asn Asp Ile Leu Glu Glu Asp Asp Asp 465 470 475 GCC AAT ACA AAC CCT TCC TCT TCC GAG CAG GCA AGC ATT GAG CTA TGT 1671 Ala Asn Thr Asn Pro Ser Ser Ser Glu Gln Ala Ser Ile Glu Leu Cys 480 485 490 490 GCT GCT GAC AAG AAC ATG CTG CAG GAG CTA GCT GAA GCT TTC AGG GTA 1719 Ala Ala Asp Lys Asn Met Leu Gln Glu Leu Ala Glu Ala Phe Arg Val 495 500 505 TCC CAC AAA GCA GAG GAG ACC CTC CCA AGC GAC GGC AGC AAT CCA GAG 1767 Ser His Lys Ala Glu Glu Thr Leu Pro Ser Asp Gly Ser Asn Pro Glu 510 515 520 ACA TGC AAT GTT CAA ATG TGC GAA GCC ATC TCT GTG CCA GAA CTC CCC 1815 Thr Cys Asn Val Gln Met Cys Glu Ala Ile Ser Val Pro Glu Leu Pro 525 530 535 540 540 TCT CAT CTC ACC TCA CAG CCA GAG GCT GGG AAC CAC TGT GAG CAG TTC 1863 Ser His Leu Thr Ser Gln Pro Glu Ala Gly Asn His Cys Glu Gln Phe 545 550 555 555 AGC ATG AGC ACA TCA GCA CCC CCT GAA CAC ATG CCA GCA GCC ACT GAA 1911 Ser Met Ser Thr Ser Ala Pro Pro Glu His Met Pro Ala Ala Thr Glu 560 565 570 AAT ATG ACA ACG CTC ATT GAG AAC ATG CCA AAG CCC ACC AAA AGC ATG 1959 Asn Met Thr Thr Leu Ile Glu Asn Met Pro Lys Pro Thr Lys Ser Met 575 580 585 CCC ATG CCC CCA GAA ATG GTG CCA ACA CCC ACA GAA AGT GTG CCA ATA 2007 Pro Met Pro Pro Glu Met Val Pro Thr Pro Thr Glu Ser Val Pro Ile 590 595 600 CCT ACA GAA GGT GTG CCA ACA CCT CCC AAA ATC ATG CCA GCA ACC CCT 2055 Pro Thr Glu Gly Val Pro Thr Pro Pro Lys Ile Met Pro Ala Thr Pro 605 610 615 620 620 GAA AGT GTG CCA ACA CTC ATG CAA AAC ACA TCT G CT CCC CTT GGA AAT 2103 Glu Ser Val Pro Thr Leu Met Gln Asn Thr Ser Ala Pro Leu Gly Asn 625 630 635 ATG CCA GCA TCC ACC AAA AGC ACA CCA AAA GCT GTA GAA CAT GTG GAT 2151 Met Pro Ala Ser Thr Lys Ser Thr Pro Lys Ala Val Glu His Val Asp 640 645 650 650 GAC ATA GCA GAG CTG TTT ATC CCT GAT CAG CCT GCA GAG CAA AAA GGG 2199 Asp Ile Ala Glu Leu Phe Ile Pro Asp Gln Pro Ala Glu Gln Lys Gly 655 660 660 665 GAT ACA GAT ATG GAA GCA GTA GAT TGA AGAC AAAGAAGAGT TCAGGCTCAT 2250 Asp Thr Asp Met Glu Ala Val Asp * 670 675 676 ATTAGACAAA CCGCAGCTCT ACTTTGTTCC TCAGATTTGT TCGTTTAAAG AAAAAAAGTG 2310 AGTGCTAAAA CACAATCATA CCTACCTGTA GACAAACATA ACCCACTTCT CTGATACCAC 2370 TCTCCAGTTT TGCTCAGCCT GCACGTTTTT CTGTCCTTCA TCTGATTAAA GCATAATATT 2430 TCCTTAATGA TAGGAAAATT CAACAAAATT ACAGTATCTT TTTTCCCAGT CATGAGTCAC 2490 CTCCTTCCAC AAAATAATTT TGAAATGCTG AAGTTACATG TTTCAAAAGT CAATGCTATT 2550 TTCATATTTC ATACATATAC TTAAAGTCTC ATAATTAATT ATTTCTATCT GATATGTTGC 2610 ATAAAAGATG GCCTACTTGG AAATGCAGAG ATCTCAATAA ATGAGGAGAAAGAGAGGTGT GATTATACAG TAAAATAGTG AAATCATCAG TGTGGTCAGC CATACCCAAA GTGAGCTGTA 2730 ATAACAAAAG GTGATTTTTT TCTTTTAAGA GTAAACTACC ATTGAGCACT ATGTGGACTG 2790 AATACCCACC AACTTTTAGC CTTACCCTGA ATTTGTCTTT TCTGGTTTGT ACTTGACAAG 2850 AGGTTTTGTT GTCGACAGAA CAATGGAAGA AGGGAGATCT TTTCTAGATT CCTCATAAAA 2910 TGACTGAAAA GAACACTAAC TTCTAAGCTG ATGGTAATTC CCAAGGTAGG GATCGCAAAC 2970 TTCCTTCTGC ATCTAAAATA AACCTGATAG TTTGTCATAC CATTTGGGAT CATGGTACCA 3030 ACAGAGACGC TCCTGACTGC TGCCAGCCCG CAAATGAGCT CAGAAGCCTG GAAGTTCAGA 3090 AGCTGGGTGC AGATTCCAGC ACTTAAGCCA GGGGCAAAAG GCAGAGACTA CACCAGGAGA 3150 TATCCCTGTG ATGATGTTTC AGGATGGTGT GATCCCTTCC AGTCACAGCC ACATGAACCT 3210 CTGTTCACTG AAGGTTCCTT CTGACACCGT TGTACATGCT GATGCAACCA GGAGAAAGGC 3270 AAGCACACTT ATCTGTCACC CTGTTCAAGT CAGTAGCCCA GAAGCAAATA TGTACCAGGA 3330 AAGCAGGAAT CTTTGATCAT TCCGGGGGCC TCTTTTTATT TCCACTTTTT TTCTCTTCTA 3390 ACCAGCTCTT CCTATCTGCT TGTCAACACA TCATTGCCTA GTAATTTCTC CCCCATGTTG 3450 GAATTGCCAA GCCAGAAGTT CTGATTTTGA CACCAAGAAC AAGGATGAGG AAGGAAGCAG 3510 GTAAG GATGT AGCTACCAAG ACAACTGGCT GGTCTCAAAA GCAAGGAGAG TAAGAAGGCC 3570 CTAGGGAAGA ACACTGAAAC AAAAATCCTG TTGGCACATT TCCTGTTTTT CATCTCCCTC 3630 CTCTGAATGC AATCTAGGTC TTTTGGATTG TGATCTGAAT AGCTTAAGAA TCCGAAAAGG 3690 GCAGTAGCGT GAGATATCCA GATGCTGGAA AGCTAAATAT TACAATCAAA CAAACCCACA 3750 GCTAGAGACA AATCACAAGC AGCCCCTGAG CAGAATCTGA TCATTTAAGC CAAAATTGAA 3810 ATACAGTTGA ATTAAGAGAA AAAATGCTAC CTTTGGCAGA ATGTGGGAGA CTGCGGTCAC 3870 ACATTCTTTC CTGCACGTCA CCCTCCAGAA GTGAGGTTTG AATCTTTGCT TTCTTCTAGC 3930 ATGTCAGCAA CCTGGCTGAG TACATCTGCA TCTGCACTGC TCATTCAGCC TTTGAGAGCT 3990 AACTCGAATA TACAGAGCAC AATTTGCAAA CGTTGGTTGT TCTAATACAG ACTAACAAGT 4050 AGCTATTAGA GCTACAGCTT CCAAATGTAG ATGCCTTATA TTAGGCTGAC AGGAGTTATT 4110 TCATGCCTTT AATCACCTGA AAGTGGATCA TTTAAGTCTT AAGCTCCTTT TATAAGTAAT 4170 GGAGATAATA GGCGTTTCTT GAGGACAAGT CATCCCCTGT TAAAACAGAT ATCCAAGACT 4230 GAAGAAAATG TCTTTCTCTT CACTGATTCT AGAATCAGCC CAAGAAAACC ATCTTAAATT 4290 TAAAATGATA AATATTAGGT GTCTAAATTA GTTAGGTAAG AAGGATCCCA CCACACATTC 4350 AGAATCAAGG TCCTAAGCAA ATCAACCAAG ACACTTTGAA AACCAAGACA CTTATGTGCC 4410 TGTGAAAATG TTAGCTTATG ACAAAATTTT ATCCTAAGGA AACAAGCTGT ATTCACTTGA 4470 TTTACTTTTA GTTCCTATTG CCTTGAGTCT GTAGAAATCT GGTTTCCATT ACAATCTCTG 4530 TTTGGTTTTT TTTTTTTGTT TTGTTTTGTT TTTAAGAGTG GTTTTAAAAC TAATGTAATT 4590 TATATAGCTT TTCTTCTCCT CTTTCTTGAT CTCATTTGAT TCTGTTGTCA GTTTTGGAAT 4650 TTCCTGCATG TAACCAACCC TTATTTTTTT AAAGTAGGCC TTCAGAATAC CTTTAAAAAA 4710 AAAAAAAAAA AAA 4723

[Brief description of the drawings]

FIG. 1 shows the protein of the present invention (1-676; ●), PD
(■), GST (▼), and no protein added (◆) are the results of tests on telencephalon.

FIG. 2: Protein of the present invention (249-676; ●), PD
(■) shows the results of GST (▲) tests on telencephalon.

FIG. 3: Protein of the present invention (1-676;）), PD
(●), GST (△), protein non-added (□) shows the results of tests on the spinal cord.

FIG. 4 shows the sequences of the DNA (1-2400) and the protein (1-676; one-letter code) of the present invention.

FIG. 5 shows the sequence of the DNA (2401 to 4723) of the present invention.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12N 1/21 C12P 21/02 H 15/09 ZNA C07K 19/00 C12P 21/02 A61K 37/02 // C07K 19/00 C12N 15/00 ZNAA (C12N 1/21 C12R 1:19) (C12N 15/09 ZNA C12R 1:19) (C12P 21/02 C12R 1:19) (58) Fields investigated (Int. Cl. ⁷ , DB name) ) BIOSIS (DIALOG) GenBank / EMBL / DDBJ WPI (DIALOG)

Claims (10)

(57) [Claims] 1. An amino acid sequence having neurite outgrowth activity
Protein represented by 1-676 of SEQ ID NO: 1, or a neurite outgrowth activity, one or several amino acids are substituted, deleted or added protein derivatives. 2. An amino acid sequence having a neurite outgrowth activity.
Protein represented by 249 to 676 of SEQ ID NO: 1, or a neurite outgrowth activity, one or several amino acids are substituted, deleted or added protein derivatives. 3. An amino acid sequence having neurite outgrowth activity
A protein represented by 1 to 676 of SEQ ID NO: 1 or
A DNA comprising a nucleic acid sequence encoding a protein derivative having one or several amino acids substituted, deleted or added, having neurite outgrowth activity . 4. An amino acid sequence having neurite outgrowth activity
Protein represented by 249 to 676 of SEQ ID NO: 1 or a neurite outgrowth activity, one or several amino acids are substituted, DNA including nucleic acid sequences encoding the deletion or addition of protein derivatives. 5. The meet DNA capable of hybridizing to the DNA under stringent conditions according to claim 3 or 4
The protein encoded by the DNA capable of hybridizing
Is DNA having neurite outgrowth activity. 6. The DNA according to claim 4, which is represented by the amino acid sequence of SEQ ID NO: 1. 7. A neurite outgrowth agent comprising the protein according to claim 1 or 2 as an active ingredient. 8. An anti-dementia agent comprising the protein according to claim 1 or 2 as an active ingredient. 9. A memory improving agent comprising the protein according to claim 1 as an active ingredient. 10. A brain function activator comprising the protein according to claim 1 or 2 as an active ingredient.