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Novel human ependymin-like protein

a technology of pendymin and ependymin, applied in the field of human ependyminlike proteins, can solve the problems of short-term memory and long-term memory effect, and achieve the effect of improving the efficiency of the promoter

Inactive Publication Date: 2004-01-22
TAKEDA PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0124] The signal peptides of the present invention can secrete various endocelluar secretory protein (or peptide) such as the protein of the present invention effectively.
[0359] For transfer of the DNA of the present invention to the host animal, it is generally advantageous to use a gene construct prepared by linking the DNA at downstream of a promoter capable of being expressed in animal cells. For example, in transferring the mice-derived DNA of the present invention, it can be linked at downstream of a promoter, which is capable of causing expression of the DNA of the present invention in various animals and is derived from animals having high homology to mice, to prepare a gene construct which can then be microinjected into the fertilized egg cell of mice, whereby a DNA transferred animal showing a high production of the protein of the present invention can be provided.

Problems solved by technology

It is known that electroshocks and anesthesia may destroy the short-term memory but have no effect on the long-term memory once established.

Method used

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  • Novel human ependymin-like protein
  • Novel human ependymin-like protein
  • Novel human ependymin-like protein

Examples

Experimental program
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Effect test

example 1

Cloning of the cDNA Coding for Human Ependymin-Like Protein

[0421] From 5 .mu.g of human placental poly(A) .sup.+RNA (Clontech), a first strand DNA was synthesized using an oligo(dT) primer having XhoI site at a terminus and SuperScript II MMLV RNase H.sup.- reverse transcriptase (Gibco BRL). Then, using E. coli DNA polymerase I and RNase H, a second strand was synthesized. In this manner, a double-stranded cDNA was obtained from the poly(A).sup.- RNA. This double-stranded DNA was treated with Pfu DNA polymerase (Stratagene) to provide blunt ends and an EcoRI adapter was added. This double-stranded DNA with the EcoRI adapter added to either end was subjected to gel filtration to remove cDNAs about 1000 bp and less and using T4 polynucleotide kinase (Takara Shuzo), the EcoRI adapters were phosphorylated. This cDNA was then digested with XhoI, and after integration with .lambda.ZAPII EcoRI-XhoI arm (Stratagene), in vitro packaging was carried out to construct a human placental cDNA lib...

example 2

Northern Hybridization of Human Tissues

[0425] A membrane filter (MTN blot, Clonetech) preblotted with 2 .mu.g of human tissue poly(A).sup.+ RNA was subjected to prehybridization in a hybridization buffer (50% formamide, 5.times.SSPE (20.times.SSPE=3.6 M sodium chloride, 0.2 M sodium phosphate (pH 7.7), 20 mM EDTA), 5.times.Denhardt's solution, 0.1% SDS, 100 .mu.g / ml heat-denatured salmon sperm DNA) at 42.degree. C.

[0426] On the other hand, the BamHI-SpeI 383 bp cDNA fragment of the cDNA coding for human ependymin-like protein as shown in FIG. 1 (the cDNA fragment having a nucleotide sequence of bases no. 643 to no. 1025 of the base sequence shown in FIG. 1), as a probe, was labeled with [.alpha.-.sup.32P]dCTP (DuPont) using Randam Primer Labeling Kit (Amersham). After 12 hours of prehybridization in a hybridization buffer (50% formamide, 5.times.SSPE, 5.times.Denhardt's solution, 0.1% SDS, 100 .mu.g / ml heat-denatured salmon sperm DNA) at 42.degree. C., hybridization was carried out ...

example 3

[0427] Cloning of cDNA Encoding Rat Ependymin-Like Protein

[0428] From 8-week-old male Sprague-Dawley rats, the whole brain was enucleated and the total RNA was prepared by the guanidine-isothiocyanate method. Then, using the oligo(dT) span column (Pharmacia), a poly(A) .sup.+RNA fraction was obtained from the total RNA prepared above. From 5 .mu.g of this poly(A) .sup.+RNA, a first strand DNA was synthesized using NotI site-terminated an oligo(dT) primer having NotI site at a terminus and SuperScript II MMLV RNase H.sup.- reverse transcriptase (Gibco BRL). Then, using E. coli DNA polymerase I, E. coli DNA ligase, and RNase H, a second strand DNA was synthesized to provide a double-stranded cDNA from the poly(A) .sup.+RNA. This double-stranded DNA was treated with T4 DNA polymerase (Gibco BRL) to prepare blunt ends and an SalI adapter was added. This double-stranded DNA with the SalI adapter added to either end was cut with NotI and subjected to gel filtration to remove cDNAs of abou...

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Abstract

The invention provides an ependymin-like protein derived a mammal, or its partial peptide or its precursor protein, or a salt thereof; a signal peptide; a DNA coding for the protein, etc. a recombinant vector; a transformant; a method of producing the protein; a pharmaceutical composition comprising the protein, etc. or DNA, an antibody against the protein, etc.; and a method for screening and a screening kit for compounds promoting the function of the protein. The protein, its partial peptide or a salt thereof has physiological activities such as a nerve-extending or nerve-regenerating activity, a gliacyte stimulating activity, and so on. The protein, etc. or the DNA is useful as a therapeutic or prophylactic agent for Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), dementia or cerebellar degeneration. The antibody against the protein, etc. can be used in the assay of the protein, etc. in a test sample. Furthermore, the protein, etc. is useful as a screening reagent for compounds or their salts capable of promoting the function of the protein.

Description

[0001] The present invention relates to a novel protein showing a physiological activity on the central nervous system such as nerve-extending activity, neuroregenerative activity, gliacyte (glial cells) stimulating activity and so on.[0002] The engram or trace of memory in the brain can be divided into two phases, namely acquisition of a short-term memory and fixation of a long-term memory. It is known that electroshocks and anesthesia may destroy the short-term memory but have no effect on the long-term memory once established. On the other hand, it is known that the biochemical reactions and structural changes leading to formation of a long-term memory as the antithesis of a short-term memory can be blocked by means of protein synthesis inhibitors such as puromycin and cycloheximide (Flexner, J. B. et al., Science, 141, 57-59 (1963); Agranoff, B. W. and Klinger, P. D., Science, 146, 952-953 (1964); Agranoff, B. W. et al., Brain Research 1, 300-309 (1966)). Therefore, it has been ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/00C07K14/475
CPCC07K14/475A61K38/00
Inventor OGI, KAZUHIROONDA, HARUO
Owner TAKEDA PHARMA CO LTD
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