WO2000007614A1 - Remedies for nerve degeneration diseases - Google Patents

Abstract

Drugs exhibiting a therapeutic effect on nerve degeneration diseases such as Parkinson's disease, Alzheimer's disease, muscular hypoplastic lateral sclerosis, Huntington's disease, brain infarction and traumatic nerve degeneration, characterized by containing a protein containing the amino acid sequence represented by SEQ ID NO:1 as the active ingredient.

Description

 Specification

 Neurodegenerative disease therapeutic agent

 Technical field

 The present invention relates to a therapeutic agent for a neurodegenerative disease.

 Background technology

 GDNF Kgl ia 1 cell line-derived neurotrophic factor (BDNF) and BDNF (brain-derived neurotrophic factor) are typical peptide-type factors that have neurotrophic factor-like effects on primary cultured dopamine neurons. ), B FGF (basic ibroblast growth factor), EGF

(epidermal growth factor). In particular, GDNF is the most potent trophic factor for dopamine neurons, and is expected to have a therapeutic effect on Parkinson's disease associated with degenerative loss of nigrostriatal dopamine neurons. ing.

 However, research on the application of the above-mentioned peptidic agents to pharmaceutical uses and the pharmacological effects by this has just begun, and sufficient data has not yet been accumulated, and these therapeutic agents and There is almost no report on its effectiveness.

If other proteins having homology to peptide factors that act as neurotrophic factors on cultured dopamine neurons, such as the above GDNF, are newly found, they will Remedies for neurodegenerative diseases including Kinson's disease Clinical application can be expected.

 In view of the above, the present inventors have studied the pharmacological activity of the above-mentioned peptide-type factors or other proteins having homology with these peptides, and have studied their pharmacological activities. I have been working hard.

 In the process, a clone isolated from the human fetal brain cDNA library, TMP-2, a transmembrane protein gene TMP-2, was newly isolated ("GEN_092E10"). From Japanese Patent Application Laid-Open No. 9-38042, U.S. Pat. No. 5,831,058) to express a specific portion of the gene, After examining the effect of the primary cultured rat midbrain dopamine neurons on the survival maintenance, it was found that this could have the effect of promoting the survival of the neurons and, consequently, the effect of treating neurodegenerative diseases. Found. The present invention has been completed based on such findings.

 Disclosure of the invention

According to the present invention, (a) expression of a protein having the amino acid sequence represented by SEQ ID NO: 1 (hereinafter referred to as “TMP-2 protein”) containing a 34-318 amino acid sequence portion And (b) amino acids in which one or more amino acids are deleted, substituted or added in the amino acid sequence of 34 to 318 of the TMP-2 protein A therapeutic agent for a neurodegenerative disease, characterized by comprising an effective amount of at least one active ingredient selected from an expression product containing a noic acid sequence portion together with a pharmaceutical carrier.

 In particular, according to the present invention, the active ingredient is a therapeutic agent for a neurodegenerative disease, wherein the active ingredient is an expression product of the amino acid sequence of 34 to 318 of the TMP-2 protein, and the active ingredient is 34 to 34 of the TMP-2 protein. — 3 18 Amino acid sequence with 6 histidine residues attached to the C-terminus and a therapeutic agent for neurodegenerative disease and the same active ingredient as TMP — 2 protein 3 4 — 3 18 amino acid sequence The present invention provides a therapeutic agent for a neurodegenerative disease, which is an MBP-TMP-2 fusion protein in which a maltose binding protein is fused to the N-terminus of the protein.

 Further, according to the present invention, it is used for the treatment of Parkinson's disease, Alchemheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, cerebral infarction, diabetic neuropathy and traumatic neurodegenerative disease. A therapeutic agent for a neurodegenerative disease is provided.

Further, according to the present invention, there are provided (a) an expression product containing a 34-318 amino acid sequence portion of a TMP-2 protein and (b) an expression product containing a 34-318 amino acid sequence of a TMP-2 protein. An effective amount of at least one active ingredient selected from an expression containing an amino acid sequence portion in which one or more amino acids have been deleted, substituted or added in the acid sequence is administered. A method for treating a neurodegenerative disease characterized by administering to a required patient is provided. You.

 In the above-mentioned treatment method, particularly preferred active ingredients include a TMP-2 protein 34-318 amino acid sequence; TMP-2 protein 34-14-318 amino acid. Histidine 6 residues linked to the C-terminus of the sequence; and

The MBP-TMP-2 fusion protein in which a maltose binding protein is fused to the N-terminal of the amino acid sequence of the TMP-2 protein at 34-318.

 Furthermore, according to the present invention, (a) an expression product containing the amino acid sequence portion of the TMP-2 protein and (b) an expression product containing the amino acid sequence portion of the TMP-2 protein; Neurodegeneration of at least one active ingredient selected from an expression containing an amino acid sequence portion in which one or more amino acids have been deleted, substituted or added in the amino acid sequence. Uses for the manufacture of a therapeutic agent for a disease can be provided.

Active ingredients particularly suitable for the use include the TMP-2 protein 34-318 amino acid sequence expression product; the TMP-2 protein 34-31 18 amino acid sequence. With 6 histidine residues bound to the C-terminus of TMP-2; and MBP-TMP-2 with a maltose-binding protein fused to the N-terminus of the 34-318 amino acid sequence of TMP-2 protein Fusion proteins can be mentioned. Abbreviations such as amino acids, peptides, nucleotide sequences, and nucleic acids in this specification are referred to in the IUPAC and IUB regulations, `` Guide for the preparation of specifications including nucleotide sequences or amino acid sequences ''. Drain ”(Japan Patent Office) and conventional symbols in the relevant field.

 Hereinafter, the TMP-2 protein used as an active ingredient in the therapeutic agent of the present invention and the TMP-2 gene used for the production thereof (hereinafter referred to as "the gene of the present invention") will be described in detail.

 As a specific example of the gene of the present invention, clone GEN-092E10 shown in Example 1 described later has

The ones deduced from the DNA array can be mentioned. The clone comprises an open reading frame of a nucleotide (nucleic acid) shown in SEQ ID NO: 2 which encodes the amino acid sequence shown in SEQ ID NO: 1; It has a base sequence. The calculated molecular weights are as shown in Examples below.

The gene of the present invention is represented by, for example, a single-stranded DNA sequence as shown in SEQ ID NO: 2, and a DNA sequence complementary to the single-stranded DNA sequence or a component containing both of them. It may be a unit. Note that the sequence of the present invention shown in SEQ ID NO: 2 is an example of one combination of codons representing each amino acid residue to be coded thereby. The gene is not limited to this, and it is of course possible to have a DNA sequence selected by combining an arbitrary codon with each amino acid residue. Selection of the codon can be performed according to a conventional method, for example, the frequency of codon usage of the host to be used can be considered [Ncl. Acids Res., 9, 43-74 (1981)]. .

 The gene of the present invention is obtained by modifying a part of the amino acid sequence shown above by substituting, deleting, or adding amino acid to amino acid sequence, and has a similar function. A DNA sequence encoding the same drug (peptide) having the following is also included. The production, modification (mutation), and the like of these peptides may occur naturally, and can be obtained by post-translational modification. In addition, these peptides can convert natural genes (the gene of the present invention) by genetic engineering techniques, for example, by using the method described in Methods in Enzymo 1 ogy, Hi, P. 350, 367-382 (1987); Id., 468 (1983);

Nucleic Acids Research, Π, p9441 (1984); Lectures on pharmacokinetics, etc. Chemical synthesis methods such as the ester method and the phosphoric acid amidite method [J. Am. Chem. Soc., 89, Ρ4801 (1967); ibid., Ρ3350 (1969) Science, 150, pi 78 (1968 ); Tet rahedron Let t., 21, p 1859 (1981); ibid., 24, p245 (1983)] The DNA can be obtained by using the obtained DNA, or by a combination of these methods.

 The gene of the present invention is isolated as follows. That is, cDNA is synthesized from mRNA extracted from various tissues such as human fetal brain, adult blood vessel, placenta, etc., and is integrated into a vector to construct a library. E. coli colonies, which have been transformed with, are formed on an agar medium, and the colonies are randomly picked up and transferred to a 96-well microplate to contain the human gene. Obtain a large number of E. coli clones.

 After culturing a small amount of each of the obtained clones, the DNA is extracted and purified, and the extracted cDNA is used as a type III enzyme to stop the elongation reaction that specifically stops the four bases according to the Deoxy-Yuichi-Mine-Yuichi method. Lines Approximately 400 base sequences are determined from the 5 'end of the gene using an automatic DNA sequencer. Based on the nucleotide sequence information thus obtained, a family gene having similarity to known animal and plant species is searched.

 The above-mentioned cDNA analysis method is described in detail by Fujiwara et al. (Riki Fujiwara, Cell Engineering, ϋ, 645-654 (1995)).

The gene of the present invention can easily and stably express the protein encoded by the gene, for example, by incorporating the gene into a vector of a microorganism and culturing the transformed microorganism. Can appear.

 More specifically, the production of the gene of the present invention can be easily carried out by a general genetic engineering technique based on the sequence information of the gene of the present invention disclosed by the present invention.

 [Molecular Cloning 2nd Ed, Cold Spring Harbor

Laboratory Press (1989); See Sequential Chemistry Laboratory Course “Gene Research Methods I, I I, I II”, edited by The Biochemical Society of Japan (1986)].

 This can be performed, for example, by using a suitable cDNA probe specific to the gene of the present invention from a human cDNA library (prepared from a suitable source cell in which each gene is expressed) according to a conventional method. Nat. Acad. ScI., USA, 78, 6613 (1981); Science, 222, 778 (1983), etc.] can be carried out by selecting a desired clone.

 In the above method, examples of the source cells include various cells and tissues expressing the target gene, and cultured cells derived therefrom.

Separation and purification of mRNA, conversion to cDNA (synthesis), and cloning can all be carried out according to conventional methods. In addition, cDNA libraries are also commercially available, and in the present invention, these cDNA libraries, for example, various cDNA libraries commercially available from Clontech Lab. Inc. Libraries can also be used. Screening of the gene of the present invention from the cDNA library can be performed according to the above-mentioned usual method. As the screening method, for example, for a protein produced by cDNA, the corresponding cDNA clone is subjected to immunoscreening using the protein-specific antibody. Selection methods, plaques, probes, colony-hybridization, etc., using probes that selectively bind to the desired DNA sequence, and combinations thereof. Can be illustrated. The probe used here is generally a DNA sequence chemically synthesized on the basis of the information on the DNA sequence of the gene of the present invention. The fragment can also be used as such a probe.

 Furthermore, based on partial amino acid sequence information of a natural extract extracted, isolated and purified from each cell and tissue, a sense primer and an antisense primer are used for screening probes. It can also be used as

 When obtaining the gene of the present invention, the PCR method

C Science, ΠΟ, 1350-1354 (1985)]. In particular, when it is difficult to obtain full-length cDNA from a library, the lace method (RACE: Rapid amplification of cDNA ends; 1 (6), 35-38 (1994)), especially the 5 'race (5' RACE) method (Fr ohman, MA, eta 1., Proc. Natl. Acad. ScI., USA., 8, 8998-9002 (1988)). The primer used when employing such a PCR method can be appropriately set based on the sequence information of the gene of the present invention already clarified by the present invention. can do.

 Isolation and purification of the amplified DNAZNA fragment can be performed according to a conventional method as described above. For example, according to gel electrophoresis, etc.

 The nucleotide sequence of the gene of the present invention or various DNA fragments obtained as described above can be determined according to a conventional method. For example, the didoxy method C Proc. Natl. Acad. ScI. USA, 74, 5463-5467 (1977)] or the Maxam-Gilbert method [Methods in Enzymology, 499 (1980)]. Such a base sequence can be easily determined using a commercially available sequence kit or the like.

 According to the use of the gene of the present invention, conventional gene recombination techniques [for example, Science, 2U, pl 431 (1984); Biochem. Biophys. Res. Comm., UO, p692 (1985);

Acad. Sci., USA, 80, P5990 (1983) and the above-cited references] can be used to obtain a recombinant protein. More specifically, the production of the protein comprises preparing a recombinant DNA capable of expressing the gene of the present invention in a host cell, introducing the gene into a host cell, transforming the cell, and culturing the transformant. It is performed by.

 Here, as the host cell, any of eukaryotes and prokaryotes can be used. The eukaryotic cells include cells such as vertebrates and yeast. Examples of the vertebrate cells include COS cells which are monkey cells [Ce11, 23, 175-182 (1981)]. And Chinese hamster ovary cells and their dihydrofolate reductase-deficient strains [Pr0N at 1. Acad. Sci. USA, 77, 4216-4220 (1980)] are commonly used. However, it is not limited to these.

Vertebrate expression vectors possess a promoter, an RNA splice site, a polyadenylation site, a transcription termination sequence, etc., which are located upstream of the gene to be normally expressed. Can be used, which may also have a replication origin if desired. Examples of the expression vector include pSV2dhfr [Mol. Cell. Biol., 丄, 854 (1981)], which has an early promoter of SV40. As eukaryotic microorganisms, yeasts are generally used. Among them, yeasts belonging to the genus Saccharomyces can be advantageously used. Expression vectors of eukaryotic microorganisms such as the yeast — For example, pAM82 having a promoter for the acid phosphatase gene [Proc. Nat 1. Acad. ScI., USA, 80, 1-5 (1983)] can be used.

 In addition, a prokaryotic gene fusion vector can be preferably used as an expression vector of the gene of the present invention. Specific examples of the vector include, for example, molecular weight Examples include pGEX-2TK and PGEX-4T-2 having a GST domain of 260 (derived from S. japonicum).

Escherichia coli and Bacillus subtilis are commonly used as prokaryotic hosts. When these are used as a host, for example, a plasmid vector that can be replicated in the host bacterium is used, and a promoter and an SD upstream of the gene are used so that the gene of the present invention can be expressed in the vector. (Shine and Dalgarno) It is preferable to use an expression plasmid to which a base sequence and an initiation codon (for example, ATG) necessary for initiation of protein synthesis have been added. Escherichia coli K12 strain and the like are often used as Escherichia coli as the above-mentioned host, and PBR322 is generally used as a vector. And improved vectors thereof are often used, but not limited thereto, and various known strains and vectors can also be used. For example, a trip motor (trp) motor, lpp flow, lac promotion A PL / PR promoter can be used.

 As a method for introducing the desired recombinant DNA thus obtained into a host cell and a method for transforming the same, various general methods can be employed. The resulting transformant can be cultured according to a conventional method, and the culture produces and expresses the target protein encoded by the gene of the present invention. As the medium used for the culture, various ones commonly used depending on the host cell employed can be appropriately selected and used, and the culture can be carried out under conditions suitable for the growth of the host cell.

 As described above, the desired recombinant protein is expressed, produced, accumulated or secreted inside, outside, or on the cell membrane of the transformant.

The above-mentioned recombinant protein may be subjected to various separation operations utilizing its physical properties, chemical properties, etc., if desired [“Biochemical Data—Yuichi Book II”, pp. 1175-1259, 1st edition, 1st edition Printing, June 23, 1980, published by Tokyo Chemical Co., Ltd .; Biochemistry, 25 (25), 8274-8277 (1986); see Eur. J. Biochem., Ill, 313-321 (1987)). Can be separated and purified. Examples of the method include, for example, ordinary reconstitution treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic shock method, ultrasonic crushing, ultrafiltration, molecular sieve Chromatography (gel filtration), adsorption chromatography, ion exchange Various liquid chromatographs such as chromatographic chromatography, affinity chromatographic, high-performance liquid chromatograph (HPLC), dialysis method, etc. Combinations and the like can be exemplified, and a particularly preferred method is an affinity chromatography using a column to which a desired protein is bound.

 It should be noted that, based on the sequence information of the gene of the present invention revealed by the present invention, for example, by utilizing the nucleotide sequence of a part or the whole of the gene, the gene in various human tissues can be used. The expression of the invention gene can be detected. This can be done in a conventional manner, for example, RT — PCR (Reverse transcribed-Polymerase chain react ion)

(Kawasaki, ES, eta 1., Amplif icat ion of RNA.In PCR Protocol, A Guide to methods and applicat ions, Academic Press, Inc., SanDiego, 2 to 27 (1991)). All can be satisfactorily performed by Northern blotting analysis (Molecular Cloning, Cold Sling Harbor Laboratory (1989)).

When the PCR method is employed, the primer used is not particularly limited as long as it is specific to the gene of the present invention capable of specifically amplifying only the gene of the present invention, and is not limited to the genetic information of the present invention. It is possible to set the sequence appropriately based on it can. Usually, this can have a partial sequence of about 15 to 30 nucleotides according to a conventional method. Preferred examples thereof are as described in Examples below.

 The expression product (recombinant protein) as an active ingredient of the therapeutic agent for neurodegenerative disease of the present invention can be suitably used as an immunizing antigen for preparing a specific antibody against the recombinant protein. By using such an antigen, desired antisera (polyclonal antibodies) and monoclonal antibodies can be obtained. The method of producing the antibody itself is well understood by those skilled in the art, and such a conventional method can be employed in the present invention [Sequence Chemistry Experimental Course “Immunobiochemical Research Method”, edited by The Biochemical Society of Japan (1986) See, for example). The antibody thus obtained can be advantageously used, for example, for purification of the expression product and measurement or identification thereof by immunological techniques.

The therapeutic agent for a neurodegenerative disease according to the present invention can be produced by using the above-described expressed product as an active ingredient in the same manner as in the production of a generally known protein agent. The active ingredients used herein include (a) an expression product containing the amino acid sequence portion of the TMP-2 protein and (b) an expression product containing the amino acid sequence portion of the TMP-2 protein. At least one selected from an expression product containing an amino acid sequence portion in which one or more amino acids have been deleted, substituted or added in the amino acid sequence This is very important. Particularly preferred such expression products include:

TMP — 2 protein 34 — 3 18 Expression of amino acid sequence portion, TMP — 2 protein 34 — 3 18 Amino acid sequence with 6 histidine residues bonded to the C-terminal It includes a recombinant protein or a fusion-type recombinant protein obtained by fusing a maltose-binding protein to the N-terminal of the amino acid sequence. In addition, the expression product as the active ingredient includes amino acids in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of 34-318 of TMP-2 protein. In addition to the expression product containing the sequence (modified amino acid sequence), a recombinant protein in which six histidine residues are linked to the C-terminus of the modified amino acid sequence, and the modified amino acid sequence fusion recombinant protein fused to maltose binding protein to the N-terminus of the sequence also, these _c are naturally encompassed is the same equivalents having the same function as the particularly preferred correct expression thereof.

Recombinant proteins useful as the protein preparation also include pharmaceutically acceptable salts thereof. Such salts include non-toxic alkali metal salts, such as sodium, potassium, lithium, calcium, magnesium, normium, ammonium, etc., prepared by methods well known in the art. And earth metal salts and ammonium salts. Further, the above salts include the active ingredient peptide of the present invention and a suitable organic acid or Non-toxic acid addition salts by reaction with inorganic acids are also included. Typical acid addition salts include, for example, hydrochloride, hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, lamate Urate, borate, benzoate, lactate, phosphate, p-toluenesulfonate (tosylate), citrate, maleate, fumarate, kohachi Examples include citrate, tartrate, sulfonate, glycolate, maleate, ascorbate, benzenesulfonate and nabsylate.

 More specifically, the therapeutic agent of the present invention is prepared into a pharmaceutical composition or a pharmaceutical preparation containing a pharmaceutically effective amount of the above-described expression together with a suitable pharmaceutical carrier (or diluent).

 Carriers that can be used in the above-mentioned pharmaceutical composition (pharmaceutical formulation) include fillers, extenders, binders, moisturizers, disintegrants, surfactants, and lubricants that are usually used according to the use form of the formulation. Diluents or excipients such as powders can be exemplified, and these are appropriately selected and used depending on the dosage unit form of the obtained preparation. Various formulations can be selected according to the purpose of treatment, and typical examples are tablets, pills, powders, solutions, suspensions, capsules, suppositories, and injections. Agents (solutions, suspensions, etc.), ointments, eye drops and the like.

Particularly preferred pharmaceutical preparations of the present invention include, for example, ordinary protein preparations. It is prepared by appropriately using various components that can be used in the present invention, for example, a stabilizer, a bactericide, a buffer, a tonicity agent, a chelating agent, a pH adjusting agent, a surfactant and the like.

 Examples of the stabilizer include human serum albumin and ordinary L-amino acids, saccharides, cellulose derivatives, and the like, which can be used alone or in combination with a surfactant or the like. Can be used. In particular, this combination may improve the stability of the active ingredient in some cases.

 The above L-amino acid is not particularly limited, and may be, for example, any of glycine, cystine, and glutamic acid.

 The above-mentioned sugars are not particularly limited. For example, monosaccharides such as glucose, mannose, galactose and fructose, sugar alcohols such as mannitol, inositol and xylitol, Polysaccharides such as disaccharides such as sucrose, maltose and lactose, dextran, hydroxypropyl starch, chondroitin sulfate, hyaluronic acid, and derivatives thereof can be used.

The surfactant is not particularly limited, and either an ionic surfactant or a nonionic surfactant can be used. For example, a polyoxyethylene render, a coal sorbin alkyl ester, and a polyoxyethylene alkyl ether can be used. System, sorby evening No acyl ester type, fatty acid glyceride type, etc. can be used.

 There is no particular limitation on the cellulose derivative, and methylcellulose, ethylcellulose, hydroxypropyl cellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and the like can be used. .

 The above sugars are added in an amount of about 1 g of the active ingredient.

It is appropriate that the concentration be in the range of about 0.1 to about 0.1 mg, preferably about 0.01 to 10 mg. The amount of the surfactant to be added is about 0.0000 mg or more per lig of the active ingredient, preferably about 0.001 to 0.1 mg. is there. The amount of human serum albumin to be added is about 0.0000 lmg or more per lg of the active ingredient, and preferably about 0.001 to 0.1 mg / l. Amino acid is preferably about 0.001 per lg of active ingredient: about L O mg. The amount of the cellulose derivative to be added is about 0.00001 mg or more per 1 g of the active ingredient, and preferably about 0.01 mg or more.

 It is appropriate to set the range to about 0.001 to 0.1 mg.

The amount of the active ingredient contained in the pharmaceutical preparation of the present invention may vary widely. It is appropriately selected from the above, but is usually in the range of about 0.0001 to 70% by weight, preferably about 0.001 to 5% by weight.

 In addition, various additives such as a buffering agent, an isotonic agent, and a chelating agent can be added to the pharmaceutical preparation of the present invention. The buffer may be boric acid, phosphoric acid, acetic acid, citric acid, ε-aminocaproic acid, glutamic acid, or a salt thereof (eg, Examples thereof include alkali metal salts such as sodium salts, calcium salts, calcium salts, and magnesium salts, and alkaline earth metal salts. Examples of the tonicity agent include sodium chloride, lithium chloride, saccharides, glycerin and the like. Examples of the chelating agent include sodium edetate and citric acid.

 The pharmaceutical preparation of the present invention can be used not only as a solution preparation, but also by lyophilizing it to make it storable, and then dissolving it in a buffer solution containing water, physiological saline, etc. before use. It can also be used after being prepared at an appropriate concentration.

The pharmaceutical preparation of the present invention may be used in the form of solid dosage forms such as tablets, pills, powders, powders, granules and capsules, and liquid dosage forms such as solutions, suspensions, emulsions, syrups and elixirs. May be prepared. These are also oral, depending on the route of administration. Preparations, parenteral preparations, nasal preparations, vaginal preparations, suppositories, sublingual preparations, ointments, etc., and can be prepared, formed or prepared according to the usual methods.

For example, when forming into tablets, lactose, sucrose, sodium chloride, glucose, urine, starch, calcium carbonate, kaolin, crystal cell, Excipients such as gay acid and calcium phosphate, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydr Binders such as roxypropylcellulose, methylcellulose, polyvinylpyrrolidone, carboxymethylcellulose sodium, carboxymethylcellulose calcium, low-substituted hydroxypropylcellulose, dried starch, sodium alginate, potassium Disintegrants such as powdered aluminum, powdered lamina, sodium hydrogencarbonate, calcium carbonate Deterioration of surfactants such as oxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, monoglyceride stearate, sucrose, stearin, cacao butter, hydrogenated oil Inhibitors, quaternary ammonium bases. Absorption enhancers, such as sodium raurylsulfate, glycerin. Humectants, such as starch, starch, lactose, kaolin, bentonite, Adsorbents such as colloidal keic acid, purified tar Lubricants such as oil, stearate, powdered boric acid and polyethylene glycol can be used.

 Furthermore, tablets should be tablets coated with a normal coating as required, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets or double or multilayer tablets. Can be.

 When formed into pills, pharmaceutical carriers such as excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, kaolin, talc, and gum arabic Powders, tragacanth powder, binders such as gelatin and ethanol, and disintegrants such as lamina lan and carten.

 The capsule preparation is prepared by mixing the active ingredient of the present invention with the above-mentioned various pharmaceutical carriers and filling the mixture into a hard gelatin capsule, a soft capsule or the like according to a conventional method.

 Liquid dosage forms for oral administration include pharmaceutically acceptable solutions, including conventional inert diluents such as water, emulsions, suspensions, syrups, elixirs, and the like. Auxiliaries such as wetting agents, emulsions and suspending agents can be included, and these are prepared according to a conventional method.

In preparing liquid dosage forms for parenteral administration, such as sterile aqueous or non-aqueous solutions, emulsions or suspensions, the diluents may be water, ethyl alcohol, pro Pyrene glycol, polyethylene glycol, ethoxylate Organic oils that can be used, such as vegetable oils such as isostearyl alcohol, polyisobutyl alcohol, polyoxyethylene sorbin fatty acid esters, and olive oil; and injectable organic esters For example, oleic acid ethyl and the like can be blended. These may further contain conventional solubilizers, buffers, wetting agents, emulsifiers, suspending agents, preservatives, dispersants, and the like. Sterilization can be carried out by, for example, a filtration operation passing through a retention filter, blending of a bactericide, irradiation treatment and heat treatment. They can also be prepared in the form of sterile solid compositions which can be dissolved in sterile water or a suitable sterilizable medium immediately before use.

 In the preparation of suppositories and vaginal preparations, pharmaceutical carriers such as polyethylene glycol, cocoa butter, higher alcohols, higher alcohol esters, gelatin and semi-synthetic glycol You can use Celide etc.

When forming into ointments such as pastes, creams, gels, etc., diluents such as white petrolatum, paraffin, glycerin, cellulose derivatives, propylene glycol Vegetable oils such as coal, polyethylene glycol, silicone, bentonite and olive oil can be used. The composition for nasal or sublingual administration can be prepared according to a conventional method using well-known standard excipients.

 The pharmaceutical preparation of the present invention may contain a coloring agent, a preservative, a flavor, a flavoring agent, a sweetening agent, and other pharmaceuticals, if necessary.

 The administration method of the above pharmaceutical preparation is not particularly limited, and is determined according to various dosage forms, patient age, gender and other conditions, degree of disease, and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules, and capsules are administered orally, and injections are used alone or mixed with normal replenishers such as glucose and amino acids to give intravenous fluids. Intramuscularly, intradermally, subcutaneously or intraperitoneally, suppositories are administered rectally, vaginal is given intravaginally, nasal is given intranasally as needed. It is administered sublingually, buccally, and ointment is topically administered transdermally.

The dosage of the pharmaceutical preparation is not particularly limited, and is appropriately selected from a wide range according to the desired therapeutic effect, administration method, treatment period, patient age, gender, and other conditions. Usually, the amount of the active ingredient should be about 0.0 lg to 10 mg, preferably about 0.1 mg to 1 mg per 1 kg of adult body weight per day. It can be administered once or several times a day. BRIEF DESCRIPTION OF THE FIGURES

 FIGS. 1 to 3 are graphs showing the effect of the protein of the present invention on the survival of nerve cells.

 BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, examples will be given to describe the present invention in further detail.

Example 1 TMP-2 gene

 (1) Cloning and DNA sequencing of the TMP-2 gene

 The mRNA extracted from human fetal brain was purchased from Clontech and used as a starting material.

 CDNA was synthesized from each of the above mRNAs, inserted into Vector Incorporated ZAP II (Stratagene), and a cDNA library was constructed (Otsuka GEN Research Institute, Otsuka Pharmaceutical Co., Ltd.)

E. coli containing a human gene on an agar medium by the in vivo excision method (In vivo excision: Short, JM, et al., Nucleic Acids Res., 16-7583-7600 (1988)). A colony was formed, the colony was picked up randomly, and an E. coli clone containing a human gene was registered in a 96-well microplate. Registered clones were stored at -80 ° C. Next, each of the registered clones was cultured overnight in 1.5 ml of LB medium, and DNA was extracted and purified using a plasmid automatic extractor P1-1100 (manufactured by Kurabo Co., Ltd.). The contaminated Escherichia coli RNA was degraded and removed by RNase treatment. Finally, the DNA was dissolved in 301, and 21 was roughly checked for the size and amount of DNA by Miguel, 7 l was used for the sequence reaction, and the remaining 21 l was used for the sequence reaction. And stored at 4 ° C as plasmid DNA.

 This was followed by a one-minute mix with Sanger et al. Using T3, Τ7 or a synthetic oligonucleotide primer.

—Sanger, F., et al., Proc. Natl. Acad. Sci., USA., 74, 5463-5467 (1977)) or dideoxy-one-one-one-one method with PCR The cycle sequence method (Carothers, AM, et al., Bio. Techniques, 7, 494-499 (1989)) was performed. They contain a small amount of plasmid DNA (approximately 0.1-0.5 g) as a template.

This is a method of performing an elongation reaction that specifically terminates four types of bases (type III).

 The sequence primer is FITC

Fluorescently labeled (fluorescein isothiocyanate) was used, and the reaction was usually performed for about 25 cycles with Taq polymerase. The PCR product is polyacrylamide urea The DNA fragments separated by gel and fluorescently labeled were sequenced using an automatic DNA sequencer and ALF ^™ DNA sequencer (Pharmacia) to sequence about 400 bases from the 5 'end of the cDNA. Were determined.

 In addition, since the 3 'untranslated region has high heterogeneity of each gene and is suitable for distinguishing individual genes, in some cases, the 3 side sequence may be used. I went there.

 The vast amount of nucleotide sequence information obtained from the DNA sequencer was transferred to a 64 bit computer, DEC340, and used for homology analysis by computer. For the holistic analysis, the data was obtained from the UWGCG FASTA program (Pearson, WR and Lipman, DJ, Proc. Natl. Acad. Sc., USA., 85, 2444-2448 (1988)). The search was performed by overnight (GenBank, EMBL) search.

 According to the above method, sequence analysis and database search of a cDNA clone arbitrarily selected from the human fetal brain cDNA library revealed that the membrane protein gene

 (transmembrane protein: Accession No .; U 19878), a clone having a highly homologous cDNA sequence (GEN-092E10) was found.

Conventionally, however, membrane protein genes have been laevis) and humans, which are transmembrane proteins containing phoristatin module (fol 1 is tat in module) and EGF¾ (epidermal growth factor doma in). It is considered to be a quality gene (accession number; U19878).

 Based on the sequence information of the above protein gene, the GEN_092E10 clone lacked 5 regions, so that the Agt10c DNA library (Human Fetal Brain 5'-STRETCH PLUS cDN A GEN-092E10 clone as a probe to obtain cDNA clones containing 5 'upstream. Isolated.

 By sequencing both strands of this cDNA clone, the sequence covering the entire coding region was revealed, and this gene was expressed in TMP-2. It was named gene.

The TMP-2 gene contains an open reading frame of 1122 bases represented by SEQ ID NO: 2, and the amino acid encoded thereby has the sequence of SEQ ID NO: 1. As shown in SEQ ID NO: 3, the nucleic acid sequence of all cDNA clones having 374 amino acid residues and having TMP-2 is 1721 bases as shown in SEQ ID NO: 3. Had become. As shown in SEQ ID NO: 3, the 5 'non-coding region was overall GC rich. There were several candidate sequences for the start codon, but according to the scanning model, the fifth ATG (SEQ ID NO:

3 6 8 — 3 7 0) was estimated to be the start codon. The stop codon was located at the nucleotide sequence No. 1490-1492 second. The poly 'adenylation' signal (ATATAAA) was located at nucleotide position 1703-1708. The calculated molecular weight of the TMP_2 gene product was 41,400 daltons.

 As described above, the membrane protein gene has a phosphoryltin module and an EGF region, and these motifs were preserved in the human gene obtained in this example.

 In addition, the TMP-2 gene is TGF-H (transforming growth factor-a; De rynck, R., eta 1., Cell, 38, 287-297 (1984)),) 3-cellulin () 3- ce 11 u 1 in; Igarashi, K. and Folkman, J., Science, 159. 1604-1607 (1993)), heparin-binding EGF-like growth factor; Hi ga siy ama, S., eta 1.,

Science, 251, 936-939 (1991)) and Schwannoma-derived growt factor (Kimura, H., et al., Nature, 348, 257-260 (1990)) and similarities at the amino acid level around the EGF region. It will play an important role in

 (2) Northern plot analysis

 The expression of mRNA of TMP-2 protein in normal human tissues is probed with a human cDNA clone labeled by the random oligonucleotide priming method. Evaluated by Southern blot.

 Northern blot analysis was performed using human MTN blots (Human Mul tiple Tissue Northern blot; Clonetech, Palo Alto, California, USA) according to the product usage. .

In other words, the GEN- 0 9 2 E 1 0 click loans PCR amplification product of ^[32 P] of - d CTP (La Ndamupurai arm de

The DNA was labeled with a DNA labeling kit (Boehringer Mannheim) to obtain a probe.

 Blotting at 42 ° C, 50% formamide Z5XSSC / 50X Denhardz solution 0.1%

Hybridization was carried out in a solution of SDS solution (containing 100 ^ g Zml denatured salmon sperm DNA). 2 After washing twice with XSSC 0.01% SDS at room temperature, then wash with 0.IX The plate was washed three times with SSCZ O. 0 5% SDS at 50 ° C for 40 minutes. The film was exposed to an X-ray film (manufactured by Kodak) at 170 ° C for 18 hours.

 As a result, high expression of TMP_2mRNA was detected in brain and prostate. The T M P — 2 genes

The size of the mRNA was about 2 kb.

Example 2 TMP-2 protein

 (1) Construction of MBP-TMP-2 fusion protein expression vector

 A region excluding the region predicted to be a transmembrane region from the coding region of the TMP_2 gene obtained in Example 1 (SEQ ID NO: 1). — Based on the DNA sequence information shown in SEQ ID NO: 3, the sequence is shown in SEQ ID NOs: 4 and 5 for subcloning the gene coding for (3-18 amino acid residue). Sequence primers A and B were made.

Primer A includes the EcoRI site, and Primer B includes the Sail site. Using both primers, PCR was performed using cDNA derived from human fetal brain mRNA to obtain a product of about 900 bases. After purifying the amplified cDNA with CHROMASPIN-400 column (manufactured by Cro-Tech), pMAL-C2 (New (England Biolabs), transformed into E. coli TB1, cloned, and the entire sequence was confirmed. The obtained fusion vector is a vector that expresses a fusion protein obtained by fusing a target TMP-2 gene product with a maltose binding protein (MBP) at the amino terminal end thereof.

 (2) Expression and purification of fusion protein

 Transform the vector prepared above into Escherichia coli (TBI) and culture the Escherichia coli containing the obtained fusion vector in an LB medium containing ampicillin at 30 ° C for 1 hour. Then, the mixture was diluted 10-fold with the same medium, cultured at 37 ° C for 2 hours, IPTG was added thereto to a concentration of 2 mM, and further cultured at 20 ° C for 10 minutes. Collect the bacteria by centrifugation, suspend them in TED containing 10% sucrose (20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM MDTT), and mix them with proteinase. A cracking kit (Sigma) was added thereto, followed by sonication, followed by ultracentrifugation to obtain a supernatant. This supernatant was applied to an amylose resin, and the column was washed thoroughly with TED containing 1% CHAPS and 0.1 M NaCl, followed by TED containing 0.1 M NaCl, and then washed. The target fusion protein (MBP-TMP-2) was eluted with TED containing 0 mM maltose.

The final eluted fraction and the fraction of each purification step are SDS -Purification and quantification were performed by PAGE. The eluted fraction was further purified using a SMART system Mono QPCI. 6Z5 column (Amersham Pharmacia Biotech) as necessary. That is, the above-mentioned eluted fraction was adsorbed to Mono QPCI. 6-5 column equilibrated with TED, and eluted with a linear concentration gradient of 0 to 0.5 MNaCl. Objective The fraction containing the fusion protein was identified by SDS-PAGE.

 The MBP for control was obtained from E. coli (TBI) transformed with pMAL-C2 vector in the same manner as the above-mentioned MBP-TMP-2 fusion protein. Purified with resin.

 (3) Construction of TMP—2—His expression vector

 A region excluding a region predicted as a transmembrane region from the coding region of the TMP-2 gene obtained in Example 1 (SEQ ID NO: 1 in the amino acid sequence shown in FIG. 1). Based on the DNA sequence information shown in SEQ ID NO: 3, subsequences of SEQ ID NOs: 6 and 7 are used to sub-clone the gene encoding 4–3 18 amino acid residues). Primers C and D of the indicated sequence were prepared.

Primer C contains N del site and Primer D contains Xhol site. Using both primers, PCR was performed using cDNA derived from human fetal brain mRNA to obtain a product of approximately 900 bases. After the resulting amplified c DNA fragment cut with N de I and X ho I, p ET 2 lb - 1 ac I q (11 ^ 1 "(^ introducing 1 ac I ^q to 611 companies made £ Ding 2 1 b The expression vector was purified.

The above p ET 21 b — lacl ^q was created as follows. That is, p ET21b (manufactured by Invitrogen) and pBluescript II were replaced with Xbal and

The fragment was digested with ApaI, and the obtained about 1 kb fragment of pET21b was subcloned into the cut pBLeuscriptII. From the obtained clone, a fragment of about 750 bp was cut out at Sph i and KpnI,

pKF19k was subcloned to the one treated with the same enzyme. Next, using a synthetic primer having the sequence shown in SEQ ID NO: 12 phosphorylated at the 5 'end, Mutan-

1 ac ^Iq (overexpressing mutant of 1 ac reblesser. Michele P. Cal os, Nature, 274, 762-765, (1978)) by Super Express Km (Yukara). After the introduction, SphI and M1uI were cut out, and sub-cloning of pET21b cut with SphI and MluI was performed to confirm the sequence. The expression vector obtained above contains a histidine tag (histidine 6) at the 34-318 amino acid residue portion of the target TMP-2 protein and its carboxyl terminus (C-terminus). This is a vector that can express a fusion protein obtained by fusing the residue.

 The expression vector obtained above was transformed into E. coli BL21 (DE3) into which pLysE plasmid had been introduced, and the obtained E. coli was suspended in LB medium. The cells were cultured at 37 ° C for 1 hour, and IPTG at a final concentration of 2 mM was added to induce 1% protein at 20 ° C.

 From the obtained culture solution, 1 ml was collected, the cells were collected, and the SDS sample 'buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 5 カ 2 mercaps) was collected. After suspending in ethanol (Ί% glycerin), heat at 100 ° C and perform 13% SDS-PAGE, then coomassie staining and anti-MBP-092 antibody. Western blotting was performed to determine the expression of the target protein.

As a result, the expression of the target protein was confirmed, but the expression level was found to be extremely small. This was presumed to be due to the fact that the N-terminal DNA sequence coding for the target protein contained many codons that were difficult to translate (low frequency) in E. coli. Therefore, in order to make the above N-terminal DNA sequence easily translatable in Escherichia coli, a high expression vector was prepared using the linker DNAs E and F shown in SEQ ID NOs: 8 and 9. .

 The amino acid sequence in the above linker sequence was the same in mice and humans, and linkers were prepared by annealing these DNAs. Its 5 'end is the site generated when cut with NdeI, and the 3' end is

It has a site that occurs when cutting at Cfr10I.

 Using this linker, we tried to exchange each cloned TMP-2 fragment with NdeI-Cfr10I fragment. However, since the linker was not successfully introduced by this method, the sequence primers P—T10 (SEQ ID NO:

After performing PCR using the sequence shown in FIG. 10), this was further converted into type I, and then re-used using Primer G (having the sequence shown in SEQ ID NO: 11) and the above-mentioned P-T10. PCR was performed.

Primer G contains an NdeI recognition sequence. The resulting PCR product was digested with N de I and X ho I, p ET 2 1 b - 1 ac I the cyclic Bok the cloning of ^q The plasmid was prepared and its DNA sequence was confirmed.As a result, it was confirmed that the plasmid contained the entire sequence of Linker E. The expression of the target protein was performed using this vector. Guidance was implemented in the same way.

 As a result, it was confirmed that the expression level of the target protein was significantly improved as compared with the case where the expression vector before reconstitution was used.

 (4) Expression and purification of the active ingredient protein of the present invention

 The expression vector reconstructed in (3) above is introduced into E. coli BL21 (DE3) strain containing pLysE plasmid, and the resulting transformant is transformed into liquid LB containing ampicillin. The cells were collected in a medium, and a part of the cells was inoculated into a liquid LB medium containing ampicillin.

 After culturing at 37 ° C until the logarithmic growth phase, IPTG was added to a final concentration of 2 mM, and the cells were cultured at 20 ° C for 1 hour. Escherichia coli was recovered from the resulting culture solution, suspended in TED containing 10% sucrose (Tris-HC1-EDTA-DTT containing 10% sucrose), and disrupted by sonication. The supernatant and the precipitate were separated by ultracentrifugation at 0.000 X g.

The precipitate is suspended in TED containing 10% sucrose, and crushing and fractionation are repeated 5 times in the same manner.The obtained precipitate is TED containing 1% CHAPS and 10% sucrose. Suspended. After sonication, the mixture was fractionated into a supernatant and a precipitate with 1000 OXg.

 The obtained supernatant is diluted with TED, developed with Ni—NTA Super—Flow column (manufactured by QIAgen), fractions containing the target protein are collected, and the MonoQ column is collected.

 (Amersham Pharmacia Biotech), collect the fractions containing the target protein, and centrifuge them.

 (Cent r i con-10, manufactured by Am icon).

 (5) Construction of TMP-2 expression vector

 TMP — 2 3 4 — 3 18 Highly expressing vector constructed in (3) above using Primer G and Primer B to subclone the gene encoding the amino acid residue. PCR was performed with one as type III.

The resulting PCR product was digested with N de I and S a 1 I, p ET 2 1 a - 1 acl the same site Bok to click b-learning the plasmid of ^q were prepared to confirm the entire nucleotide sequence. The recombinant protein obtained by this vector does not contain any tag other than the 34-318 amino acid residue of TMP-2.

 (6) Expression and purification

Using the vector obtained in the above (5), the target protein was expressed in the same manner as in the above (4). E. coli obtained The cells were suspended in TED containing 10% sucrose, added with protease inhibitor, disrupted by sonication, and then ultracentrifuged to obtain a supernatant and a precipitate. The resulting suspension was subjected to the same suspension and ultracentrifugation. To the third and fourth supernatants, a saturated ammonium sulfate solution dissolved in TED was added so as to obtain a 25% saturation, and the mixture was allowed to stand at 4 ° C, and then centrifuged to obtain a precipitate. The precipitate was suspended in TED containing protease inhibitor and dialyzed against the same buffer. The precipitate generated during dialysis was removed by centrifugation, and the target protein was purified from the supernatant by the following procedure.

 That is, the supernatant was applied to a Q-Sepharose FF column equilibrated with TED, and eluted with TED containing NaCl. NaC1 was added to a final concentration of 2 M, and applied to octylous cell mouth fins (Octy celluloiine, manufactured by Seikagaku Corporation).

 The target TMP-2 recombinant protein was mainly recovered in the non-adsorbed fraction. Ammonium sulfate was added to this fraction to make 50% saturated ammonium sulfate solution, and TMP-2 was precipitated. This precipitate

After suspending in T ED and desalting, the column

(Amersham Pharmacia Biotech) to further purify. For elution, a linear concentration gradient of 0 to 0.3 M NaCl was followed by washing with 0.3 MNaCl for a while. This was done with a linear concentration gradient of 0.3-0.5 M NaCl.

 Thus, TMP—2 was recovered with high purity in the 0.3-0.5 M NaCl fraction. This fraction was used as the final purified fraction. Concentrations were determined on SDS-PAGE, using serum albumin as an internal standard. The purified TMP-2 was frozen in liquid nitrogen and stored at -80 ° C.

 (7) Isolation and culture of brain nerve cells

 The whole brain was aseptically removed from a 15-day-old fetal SD rat and the ventral midbrain was cut off. 0.25% trypsin, 0.02% after shredding

The cells were incubated at 37 ° C for 20 minutes in phosphate buffered saline (PBS) containing DNase and treated with the enzyme. After adding fetal calf serum to stop the enzyme reaction, the procedure of sucking up and discharging the cell fluid with a pipette fitted with a plastic tip was repeated three times to disperse the cells. The cell solution was filtered through a filter in which two lens papers were stacked to remove undigested tissue fragments, and centrifuged at 100 rpm for 5 minutes. The cells were washed using DME MZ F12 medium (manufactured by Gibco) and coated with poly-L-lysine containing DME MZF12 medium containing 10% fetal bovine serum. Cells in final plate 3.1 x 10 ⁵ cells Z cm ² Sowed to become

 (8) Treatment with the active ingredient protein of the present invention

 After culturing the above cells for 24 hours, the culture solution was replaced with DMEMF 12 containing 1% N 2 additive (N2 Supplement, manufactured by GI BC0), and prepared in each of (2) and (4) above. The active ingredient protein of the present invention was added at a concentration of 20, 200 and 400 ng Zm 1 (group of the present invention).

 For comparison, carbohydrate (MBP group) supplemented with MBP (400 ng / m1) and fusion protein (40 ng Zml) heated in a boiling water bath for 5 minutes were added (boiling fusion protein group). I went.

 (9) Tyrosine hydroxylase immunohistochemistry

 After culturing the cells (culture solution) of each group prepared in the above (8) for 72 hours, the cells were fixed by leaving them at room temperature for 15 minutes using 4% paraformaldehyde dissolved in PBS. The membrane was made permeable using% Triton X100 ZPBS.

In order to prevent non-specific binding of the antibody, the cells were incubated for 1 hour with PBS containing 10% goat serum, and then the anti-tyrosine hydroxylase polyclonal antibody (CHEMIC0N; The cells were incubated for 16 hours at 4 ° C using a 1: 1000 dilution in PBS. After removing the antibody solution, remove the cells The cells were washed with PBS, peroxidase-labeled dextran polymer-conjugated goat anti-rabbit immunoglobulin (manufactured by DAK0) was added, and the mixture was incubated at room temperature for 1 hour.

 The detection of tyrosine hydroxylase-positive cells was based on the presence or absence of a color reaction using diaminobenzidine as a substrate. The survival of dopamine neurons was evaluated using the number of cells that are positive for synthase hydroxylase.

 (10) Counting tyrosine hydroxylase-positive cells

The number of tyrosine hydroxylase-positive cells in a field, which is equivalent to 10% of the area of a well per well, is counted for 11 fields (magnification: X100) The measured value was converted to the number of cells per cm ² .

(11) Effect of the active ingredient protein of the present invention on the survival of primary cultured rat midbrain dopamine neurons The results are shown in Figs. 1 and 2 (vertical axis: number of tyrosine hydroxylase-positive cells (cells / cm ² ), horizontal axis: each group).

 In FIG. 1, each group is as follows, and n indicates the number of jewels.

Control group: control group to which no additives were added (n = 8)

MBP group: MBPAOOng Zml added group (n = 8) Boiling fusion protein group: M heated for 5 minutes in a boiling water bath BP — TMP — 2 fusion protein 400 ng / m 1 added group (n = 8)

Group of the present invention: group to which 400 ng ml of MBP-TMP-2 fusion protein was added (n = 8)

 In the figure, * indicates no significant difference from the control group, and ** indicates significant difference from the control group (P <0.05, due to Dunnett's test).

 From Fig. 1, it is clear that in the present invention group to which the MBP-TMP-2 fusion protein was added, the survival of dopamine neurons was promoted (suppression of cell death) depending on the fusion protein. is there. This effect was significantly lower than p <0.05

 (Due to Dunnett's st e st).

 No significant difference was observed in the MBP group from the control group. In addition, in the boiling fusion protein group, the effect of maintaining survival on dopamine neurons disappeared.

 In FIG. 2, each group is as follows, and n represents the number of jewels.

Control group: control group without any additives (n = 6)

Group of the present invention: TMP—2—His fusion protein to which 0.1, 1 and lOng Zml were added, respectively (n = 6). , * * Is significantly different from the control group (p <0.05, according to Dunnett's test), and *** is significantly different from the control group (p <0.01). , Dunnett's test).

 From FIG. 2, it is clear that in the group of the present invention to which the TMP—2—His fusion protein was added, the survival of dopamine neurons was promoted depending on the concentration of the fusion protein. . This effect was significantly different from the control group with p <0.05 (by the Dun it's test) at 1 ng Zm1 of the fusion protein and at p <0.05 with the fusion protein lOng Zm1. A significant difference of 0.01 (according to Dunne 11's test) was shown.

 From the above, both the MBP-TMP-2 fusion protein and the TMP-2-His fusion protein can exert a significant neuronal cell survival promoting effect, and this effect is exerted on the TMP-2 protein. It is clear that they are based.

 Further, in the test shown in FIG. 2 above, the active ingredient protein (TMP) of the present invention prepared in the above (6) was replaced with the active ingredient protein of the present invention (TMP-2−His) prepared in the above (4). — 2 of 3 4 — 3 1 8 Amino acid sequence power, partial protein, indicated as “TMP — 2” in the table)

The same test was repeated except that they were added at 0.1 and lng / ml, respectively. The obtained results are shown in FIG. 3 as in FIG. In FIG. 3, * indicates that there is no significant difference from the control group, ** indicates that there is a significant difference from the control group (p <0.05, according to Dunnett's test), and *** Indicates a significant difference from the control group (p <0.01, according to Dunnett's test).

 FIG. 3 also shows that in the group of the present invention to which a specific partial protein of TMP-2 was added, the survival of tyrosine hydroxylase-positive cells was promoted depending on the concentration of the protein. The results clearly indicate that certain parts of TMP-2 can have a neuronal cell survival promoting effect.

 Possible industrial use

 According to the present invention, it is possible to provide a therapeutic agent for a neurodegenerative disease containing a recombinant protein that functions as a neurotrophic factor as an active ingredient, by regarding a factor having a nerve cell survival promoting effect as a neurotrophic factor. It is effective in treating neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, octantin's disease, and cerebral infarction. Diabetic neuropathy, traumatic neurodegenerative disease, etc. .

Claims

The scope of the claims 1. (a) an expression product containing the amino acid sequence portion of the amino acid sequence of the protein having the amino acid sequence represented by SEQ ID NO: 1; (b) Amino acid in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of 34 to 318 amino acid sequence represented by SEQ ID NO: 1 A therapeutic agent for a neurodegenerative disease, comprising an effective amount of at least one active ingredient selected from an expression product containing a sequence portion together with a pharmaceutical carrier.  2. The therapeutic agent for neurodegenerative disease according to claim 1, wherein the active ingredient is an expression product of the amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1 which is an expression of 34-318 amino acid sequence.  3. The active ingredient is a protein having the amino acid sequence represented by SEQ ID NO: 1 in which 34 residues of histidine are bound to the C-terminal of the amino acid sequence. 3. The therapeutic agent for a neurodegenerative disease according to item 1.  4. The active ingredient is a fusion protein obtained by fusing a maltose binding protein to the N-terminus of the amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1. Item 7. The therapeutic agent for a neurodegenerative disease according to Item 1. 5. Parkinson's disease, Alzheimer's disease, myotonic lateral sclerosis, Huntington's disease, cerebral infarction, diabetic nerve The therapeutic agent for a neurodegenerative disease according to claim 1, which is used for treating neuropathy and traumatic neurodegenerative disease. (a) an expression product containing the amino acid sequence portion of the protein having the amino acid sequence represented by SEQ ID NO: 1; and (b) an amino acid represented by SEQ ID NO: 1; At least one selected from an expression product containing an amino acid sequence portion in which one or more amino acids are deleted, substituted or added in the amino acid sequence of 34 to 318 amino acid sequence of the sequence A method for treating a neurodegenerative disease, comprising administering an effective amount of at least one active ingredient to a patient in need of treatment.  7. The method for treating a neurodegenerative disease according to claim 6, wherein the active ingredient is an expression product of the amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1 which is an expression of 34-318 amino acid sequence. 7. The method according to claim 6, wherein the active ingredient is a protein having the amino acid sequence represented by SEQ ID NO: 1 in which 34 residues of histidine are bonded to the C-terminal of the amino acid sequence. The method for treating a neurodegenerative disease according to the above. The active ingredient is a fusion protein obtained by fusing a maltose binding protein to the N-terminal of the amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1. 7. The method for treating a neurodegenerative disease according to item 6. 0. Parkinson's disease, Alzheimer's disease, Muscle dysfunction 7. The method for treating a neurodegenerative disease according to claim 6, which is used for treating lateral sclerosis, Huntington's disease, cerebral infarction, diabetic neuropathy and traumatic neurodegenerative disease.  1. (a) an expression product containing the amino acid sequence portion of the amino acid sequence represented by the amino acid sequence represented by SEQ ID NO: 1; and (b) an amino acid sequence represented by the amino acid sequence represented by SEQ ID NO: 1 In the amino acid sequence of the amino acid sequence of the amino acid sequence, one or more amino acids are deleted, substituted, or added in the amino acid sequence. Use of at least one active ingredient for producing a therapeutic agent for a neurodegenerative disease. 2. The use according to claim 11, wherein the active ingredient is an expression product of the amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1.  3. The active ingredient is a protein having an amino acid sequence represented by SEQ ID NO: 1 wherein 34 histidine 6 residues are bonded to the C-terminal of the amino acid sequence. Use as described in 1.  4. The active ingredient is a fusion protein obtained by fusing a maltose binding protein to the N-terminal of the amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1. Use as described in 1. 5. Parkinson's disease, Alzheimer's disease, muscle dysfunction Use of the expression product of claim 11 for the manufacture of a therapeutic agent for lateral sclerosis, Huntington's disease, cerebral infarction, diabetic neuropathy and traumatic neurodegenerative disease.