EA003734B1 - Neurotrophic factors - Google Patents

Abstract

1. An isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of: a) nucleotides 405 - 719 of SEQ ID NO. 1; b) a nucleotide sequence that hybridizes specifically to a complement sequence of nucleotides 405 - 719 of SEQ ID NO. 1 under high stringency conditions; wherein the nucleotide sequence codes for a neublastin polypeptide comprising the following: (i) seven conserved cysteine residues at positions 8, 35, 39, 72, 73, 101 and 103 when numbered in accordance with SEQ ID NO. 2; (ii) amino acid residues as follows: C at position 8, L at position 10, V at position 17, L at position 20, G at position 21, L at position 22, G at position 23, E at position 28, F at position 32, R at position 33, F at position 34, C at position 35, G at position 37, C at position 39, C at position 72, C at position 73, R at position 74, P at position 75, F at position 83, D at position 85, S at position 97, A at position 98, C at position 101 and C at position 103, each when numbered in accordance with SEQ ID NO. 2; (iii) an LGLG repeat, an FRFC motif, a QPCCRP motif, and a SATACGC motif; and (iv) an amino acid sequence having at least 90% sequence identity to AA1 - AA105 of SEQ ID NO. 2. 2. A nucleic acid comprising a nucleic acid sequence that is complementary to the nucleic acid of claim 1. 3. A vector comprising the nucleic acid of claim 1. 4. The vector of claim 3, wherein said vector is an expression vector. 5. A strain of cells transformed in vitro with the vector of claim 3 or 4. 6. The strain of claim 5, wherein said cells are selected from the group consisting of mammalian cells, fungal cells, yeast cells, insect cells and bacterial cells.] 7. The strain of claim 6, wherein said cells are Chinese hamster ovary cells. 8. The strain of claim 6, wherein said cells are cells derived from the mammalian central nervous system. 9. A nucleic acid molecule encoding a neublastin polypeptide, the nucleic acid molecule having the sequence set forth in SEQ ID NO. 29 or 30. 10. A vector comprising a nucleic acid of claim 9. 11. The vector of claim 10, wherein said vector is an expression vector. 12. A strain of cells transformed in vitro with the vector of claim 10 or 11. 13. The strain of claim 12, wherein said cells are bacterial cells. 14. An isolated neublastin polypeptide comprising the following: (a) seven conserved cysteine residues at positions 8, 35, 39, 72, 73, 101, and 103 when numbered in accordance with SEQ ID NO. 2; (b) amino acid residues as follows: C at position 8, L at position 10, V at position 17, L at position 20, G at position 21, L at position 22, G at position 23, E at position 28, F at position 32, R at position 33, F at position 34, C at position 35, G at position 37, C at position 39, C at position 72, C at position 73, R at position 74, P at position 75, F at position 83, D at position 85, S at position 97, A at position 98, C at position 101 and C at position 103, each when numbered in accordance with SEQ ID NO. 2; (c) an LGLG repeat, an FRFC motif, a QPCCRP motif, and a SATACGC motif; and (d) an amino acid sequence comprising at least 90% sequence identity to AA1 - AA105 of SEQ ID NO. 2. 15. The polypeptide of claim 14, wherein said polypeptide is glycosylated. 16. The polypeptide of claim 14, wherein said polypeptide is coded for by a nucleic acid of claim 1 or 2. 17. The polypeptide of any one of claims 14-16, wherein the polypeptide has a C-terminal amino acid sequence as set forth in AA72-AA105 of SEQ ID NO. 2. 18. The polypeptide of any one of claims 14-16, wherein the polypeptide has a C-terminal amino acid sequence as set forth in AA41-AA105 of SEQ ID NO. 2. 19. The polypeptide of any one of claims 14-16, wherein the conservative amino acid substitutions represent less than 10% of the total number of residues in the polypeptide. 20. The polypeptide of claim 19, wherein the conservative amino acid substitutions represent less than 2% of the polypeptide. 21. The polypeptide of claim 19 wherein the conservative amino acid substitutions represent a single amino acid substitution in the mature peptide sequence, wherein the both the substituted and replacement amino acid are non-cyclic. 22. A neublastin peptide having any of the following sequences: GPGSRARAAGARGC (AA 30-43 of SEQ ID NO. 9); LGHRSDELVRFRFC (AA 57-70 of SEQ ID NO. 9); CRRARSPHDLSL (AA 74-85 of SEQ ID NO. 9); LRPPPGSRPVSQPC (AA 94-107 of SEQ ID NO. 9); STWRTVDRLSATAC (AA 123-136 of SEQ ID NO. 9); CRLRSQLVPVRALGLGHRSDELVRFRFC (AA 43-70 of SEQ ID NO. 9); CRRARSPHDLSLASLLGAGALRPPPGSRPVSQPC(AA 74-107 of SEQ ID NO. 9); CRPTRYEAVSFMDVNSTWRTVDRLSATAC (AA 108-136 of SEQ ID NO. 9); CRPTRYEAVSFMDVNST (AA 108-124 of SEQ ID NO. 9); and ALRPPPGSRPVSQPC (AA 93-107 of SEQ ID NO. 9). 23. A composition comprising the polypeptide of any one of claims 14-22 and a pharmaceutically acceptable carrier. 24. A method of administering the polypeptide of any one of claims 14-16, comprising the step of delivering said polypeptide to an in vitro cell culture or in vivo to a mammal. 25. The method of claim 24, wherein said administration comprises systemic administration. 26. The method of claim 24, wherein said mammal is afflicted with a condition selected from the group consisting of cerebral ischaemic neuronal damage, traumatic brain injury, peripheral neuropathy, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, and memory impairment. 27. The method of claim 24, wherein said mammal is afflicted with a neuronal disorder of the peripheral nervous system, the medulla, or the spinal cord. 28. A use of the nucleic acid of claim 1 or 2 for the manufacture of a medicament for treatment of a neurodegenerative disease or disorder in an animal. 29. A use of the neublastin polypeptide of any one of claims 14-16 for the manufacture of a medicament for treatment of a neurodegenerative disease or disorder in an animal. 30. An antibody that specifically binds to any one of the polypeptides of any one of claims 14-16. 31. The antibody of claim 30, wherein said antibody is a monoclonal antibody.32. A method of determining whether a neurodegenerative disease or disorder in an animal is associated with an altered activity in a neublastin neurotrophic factor polypeptide, said method comprising the steps of: (a) contacting a biological sample from said animal with the antibody of claim 30 or 31, (b) determining whether an immune complex forms between said antibody and said protein, (c) comparing a level of said immune complex that forms in said sample with a level of said immune complex that forms in a corresponding biological sample from a patient lacking said neural condition, and (d) determining from said comparison whether said disease or disorder is associated with said altered level of activity in said neublastin neurotrophic factor polypeptide. 33. A strain of cells comprising an exogenous regulatory sequence cloned in operative association with an endogenous neublastin gene, said neublastin gene being capable of encoding the polypeptide of claim 14. 34. A method for producing a neublastin polypeptide of any one of claims 14-22, the method comprising growing cells of the strain of any one of claims 5-8, 12, 13 and 33 under conditions which allow expression of the polypeptide. 35. The method of claim 34, further comprising isolating the resulting polypeptide. 36. An isolated nucleic acid sequence comprising nucleotides 721-865 of SEQ ID NO. 1. 37. An antibody which specifically binds to any of the peptides of claim 22. 38. A kit comprising, in one or more containers, a substance selected from the group consisting of: (a) neublastin polypeptide according to any one of claims 14-22; (b) an antibody of any one of claims 30, 31 and 37; (c) one or more nucleic acid probes capable of hybridizing to neublastin RNA; or (d) at least one pair of nucleic acid primers capable of priming amplification of at least a portion of a neublastin gene. 39. A method of diagnosing or screening for the presence of or a predisposition for developing a disease or disorder characterized by an aberrant level of a neublastin polypeptide according to any one of claims 14-22, in a subject comprising measuring the level of said neublastin polypeptide, RNA encoding the neublastin polypeptide, or functional activity of the neublastin polypeptide in a sample derived from the subject in which an increase or decrease in the level of the neublastin polypeptide, neublastin RNA, or functional activity of neublastin polypeptide in the sample, relative to the level of the neublastin polypeptide, neublastin RNA or functional activity of neublastin found in an analogous sample not having the disease or disorder or a predisposition for developing the disease or disorder, indicates the presence of the disease or disorder or a predisposition for developing the disease or disorder. 40. A method for screening a purified neublastin polypeptide according to any one of claims 14-22, or derivative of fragment thereof, or a modulator of the activity of the foregoing, for activity in treating or preventing a disease, the method comprising: (a) measuring alterations in the phenotype, genotype, behavior, survival or proliferation of cells from a cell line or test animal, said cells or animal being derived from or display characteristics associated with the disease, and said cells or animals having been contacted with or administered with the neublastin polypeptide, derivative, fragment, or modulator, and (b) comparing said alterations with the phenotype, genotype, behavior, survival or proliferation in cells or animals not so contacted with or administered with the neublastin polypeptide, derivative, fragment, or modulator. 41. A method of treating peripheral neuropathies in a mammal, which method comprises administering a therapeutically effective amount of a neublastin polypeptide according to any one of claims 14-22, wherein said peripheral neuropathy is

Description

The present invention relates to polypeptides of neurotrophic factors, nucleic acids encoding the polypeptides of neurotrophic factors, and antibodies that specifically bind to neurotrophic factors.

BACKGROUND OF THE INVENTION

Neurotrophic factors are naturally occurring proteins that contribute to survival, provide phenotypic differentiation, prevent degeneration and enhance the activity of nerve cells and tissues. Neurotrophic factors are isolated from nervous tissue and from tissue different from the nervous but innervated nervous system and subdivided into functionally and structurally close groups, also called families, superfamilies, or subfamilies. Among the superfamilies of neurotrophic factors, there are superfamilies of fibroblast growth factor, neurotrophin, and transforming growth factor-β (TSE-β). Certain types of neurotrophic factors differ in physical structure, in their interaction with their complex receptors and in their effect on various types of nerve cells. According to the classification, the TSE-β superfamily (Makkadie, e! A1., Tgepbk ίη Ce11 одуο1odu, 1994, 4, 172-178) includes ligands that are neurotrophic factors derived from the glial cell line (ΟΌΝΕ; XV Ο 93/06116, included here by reference), to which ΟΌΝΕ refers, perseffin (P8P; MiBap! e! a1., No. Igop, 1998, 20, 245-253, included here by reference) and neurturin (ΝΤΝ; XVO 97/08196, included here by reference). A common property of subfamily ли ligands is their ability to induce signal transduction through the BET receptor tyrosine kinase. These three ligands of the subfamily ΟΌΝΡ differ in their relative affinity for the family of neurotrophic receptors, CEB receptors.

Since neurotrophic factors affect the nervous tissue, there is a need for identification and characterization of additional neurotrophic factors for the diagnosis and treatment of disorders of the nervous system.

SUMMARY OF THE INVENTION

This invention relates to a new neurotrophic factor, referred to herein as neublastin or ΝΒΝ. Neublastin is assigned to the subfamily ΟΌΝΕ since it contains regions homologous to those of other ligands ΟΌΝΕ (see Tables 3 and 4 below) and is able to interact with BET (see, for example, Ayakktep e! A1., Mo1. Ce11. Nei ^ Exx. (1999, 13, 313-325), neublastin is a new and unique neurotrophic factor. Unlike other ligands ΟΌΝΕ, neublastin exhibits a high affinity for the CEB-3-BET receptor complex and has unique regions in the amino acid sequence.

The term neublastin polypeptide, as used herein, means a polypeptide that has neurotrophic activity (for example, as described in examples 6, 7, 8 and 9), and includes those polypeptides that have an amino acid sequence of at least 70% homologous to human neublastin polypeptides represented by the amino acid sequences AK- ₉₅ -AK ₁₀₅ in 8EO ΙΌ ΝΟ: 2, AK! -AK ₁₀₅ in 5EO GO ΝΟ: 2, LK _-97 -LK ₁₄₀ in 5EO GO ΝΟ: 4 , AK _-41 -AK ₁₄₀ to 8Er GO ΝΟ: 4, (pro), AK! -AK ₁₄₀ to 5EO GO ΝΟ: 4, LK _-80 -LK ₁₄₀ to 5EO GO ΝΟ: 9 (prep type), AK _-41 -AK ₁₄₀ in 5EO GO ΝΟ: 9 (pro), AK ^ AK ^ in 8Er GO ΝΟ: 5 (mature, 140 LK), AK ₁ -LK ₁₁₆ in 5EO GO ΝΟ: 6 (mature , 116 LK), AK _1- LK ₁₁₃ in 5EO GO ΝΟ: 7 (mature, 113 LK), AK ^ AK ^ in 5EO GO ΝΟ: 10 (mature, 140 LK), AK ₁ -AK ₁₁₆ in 5EO GO ΝΟ: 11 (mature, 116 BOS), AK! -LK ₁₁₃ in 5EO GO ΝΟ: 12 (mature, 113 BOS), and their variants and derivatives. In addition, this invention contemplates polypeptides that have an amino acid sequence that is at least 70% homologous to mouse neublastin polypeptides represented by AK1LK ₂₂₄ in 5EO GO ΝΟ: 16.

Preferably, the C-terminal sequence of the neublastin polypeptides described above has an amino acid sequence that is represented by AK ₇₂ -AK _{£ 05} in 5EO GO ΝΟ: 2 (i.e., AK ₁₀₇ -AK ₁₄₀ in 5EO GO ΝΟ: 9), more preferably AK ₄₁ -AK ₁₀₅ in 8EO GO ΝΟ: 2 (i.e., AK ₇₆ -AK ₁₄₀ in 5EO GO ΝΟ: 9) or the amino acid sequence represented by AK ₁₉₁ -AK ₂₂₄ in 5EO GO ΝΟ: 16.

It is also preferred that 7 conserved Suk residues that are characteristic of the семейства family and the TSE-beta superfamily are retained in the neublastin polypeptide.

Preferably, the neublastin polypeptide has an amino acid sequence more than 85% homologous, more preferably more than 95% homologous to the sequences mentioned above (i.e., LK _-95 -LK ₁₀₅ in 5EO GO ΝΟ: 2, AK ^ -AK ^ in 5EO GO ΝΟ: 2, AK _-97 -AK ₁₄₀ to 5EO GO ΝΟ: 4, AK _G AK ₁₄₀ to 5EO GO ΝΟ: 4, AK _-41 -AK ₁₄₀ to 5EO GO 4: 4, AK- _80- AK1 ₄₀ to 8EO GO ΝΟ: 9 (wild type prepro), AK _-4 1-AK1 ₄₀ in 8EO GO ΝΟ: 9 (pro), AK! -AK ₁₄₀ in 5EO GO ΝΟ: 5 (mature, 140 LK), AK ₁ -LK ₁₁₆ in 5EO GO ΝΟ: 6 (mature, 116 LK), AK _1- LK ₁₁₃ in 5EO GO ΝΟ: 7 (mature, 113 LK), AK! -AK ₁₄₀ in 5EO GO ΝΟ: 10 (mature, 140 LK), AK ₁ -ac ₁₁₆ 5EO GO Ν : 11 (mature, 116 LC), AK ₁ -LK ₁₁₃ 5EO GO ΝΟ: 12 (mature, 113 LC), and AK AK _{224 g} 5EO GO ΝΟ: 16.

Neublastin nucleic acid, as used herein, means a polynucleotide that encodes a neublastin polypeptide. Accordingly, an isolated neublastin nucleic acid is a polynucleotide molecule having an open reading frame of nucleotide codons that carries information or encodes a neublastin polypeptide by the action of the corresponding components necessary for translation. The neublastin nucleic acids of the invention may be RNA or DNA, for example, genomic DNA or DNA that is complementary to neublastin mRNA and / or transcribed on that mRNA (cDNA). Thus, the neublastin nucleic acid of the invention also includes polynucleotide molecules which, under very stringent hybridization conditions, specifically hybridize to a polynucleotide that encodes a neublastin polypeptide. This invention also relates to nucleic acid primers that are useful for identifying, isolating and amplifying polynucleotides that encode neublastin polypeptides or fragments thereof. In some embodiments of the invention, some of these primers are neublastin-specific probes suitable for hybridization with neublastin nucleic acid, but not with nucleic acids encoding other members of the ΟΌΝΤ family. The terms specific, specific, or specific mean the ability to hybridize with a neublastin nucleic acid and the inability to hybridize to non-neublastin nucleic acids, including the inability to hybridize to nucleic acids that uniquely encode ΟΌΝΡ ligands (e.g., ΟΌΝΡ, perseffin and neurturin).

In another embodiment, the neublastin nucleic acid of the invention is an acid that is identified as a nucleic acid complementary to a polynucleotide that encodes a neublastin polypeptide, either because it has a complementary nucleic acid sequence, or by demonstrating that it hybridizes specifically with very stringent hybridization conditions polynucleotide that encodes neublastin. Specific nucleic acids of the invention include, but are not limited to, the nucleic acid sequences shown herein, designated 8EO ΙΌ N0: 1, 8EC ΙΌ N0: 3, 8EC ΙΌ N0: 8, 8EC ΙΌ N0: 13, 8EC ΙΌ N0: 14, 8EC ΙΌ N0: 15 , 8ES ΙΌ N0: 29 and 8E0 ΙΌ N0: 30, as well as primers 8E0 ΙΌ N0: 17-28, 31 and 32. The nucleic acid of the invention also includes a unique region or fragment of a neublastin nucleic acid, including, but not limited to the nucleic acid copes shown in FIG. 8.

The neublastin nucleic acids of the invention can be used to express a neublastin polypeptide, for example, by expressing a neublastin polypeptide ίη νίίτο, or by introducing a neublastin nucleic acid into an animal to express ίη νίνο. Neublastin nucleic acids may be included in a vector nucleic acid, for example, an expression vector or a cloning vector. Neublastin nucleic acid may, but is not necessary, be maintained, reproduced, transmitted, or expressed as part of a vector nucleic acid. A recombinant expression vector containing a polynucleotide sequence of neublastin can be introduced and / or stored within the cell. Prokaryotic cells may serve as host cells for the neublastin vector. Alternatively, a neublastin nucleic acid may be introduced into a eukaryotic cell, for example a eukaryotic cell, which contains a suitable apparatus for post-translational processing of the polypeptide into a mature protein, and / or a suitable apparatus for secretion of the polypeptide into the extracellular environment of the cell.

The invention also characterizes a neublastin neurotrophic factor, neublastin. Neublastin may be in the form of a polypeptide or may be a multimer of two or more neublastin polypeptides, for example in the form of a neublastin dimer. Neublastin polypeptides are associated into multimers using intermolecular structural bonds known to those skilled in the art, including but not limited to cysteine-cysteine interactions, disulfide bonds, and non-covalent interactions. Specific neublastin polypeptides include, without limitation, the amino acid sequences claimed herein and designated 8EC ΙΌ N0: 2, 8E0 ΙΌ N0: 4, 8 EC) ΙΌ N0: 5, 8 EC) ΙΌ N0: 6, 8 EC) ΙΌ N0: 7, 8EC) ΙΌ N0: 9, 8EC) ΙΌ N0: 10, 8EC) ΙΌ N0: 11, 8EC) ΙΌ N0: 12 and 8EC) ΙΌ N0: 16.

The neublastin polypeptide of the invention is useful for treating a neuron defect, including, but not limited to, affected neurons and injured neurons. Peripheral nerves that experience trauma include, but are not limited to, cranial nerves originating from the medulla oblongata, or spinal nerves. Neublastin polypeptides are useful for treating a neurodegenerative disease, for example, damage to neurons due to cerebral ischemia, neuropathy, for example peripheral neuropathy, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS). In addition, it is contemplated to use neublastin polypeptides for treating impaired memory, for example, memory impairment associated with dementia.

Brief Description of Drawings

FIG. 1 is a photographic image of two Northern blots analyzed using ³² P labeled neublastin cDNA probes, which compare the relative levels of neublastin gene expression in different types of adult tissue (panel A) and in different areas of the adult brain (panel B).

FIG. 2 is a photograph of a Northern blot analyzed using a ³² P labeled neublastin cDNA probe, which compares the amount of neublastin cDNA expressed in an untransfected H1B5 cell line with the amount of neublastin cDNA expressed in a cell line transfected with neublastin cDNA and a cell line, transfected ΟΌΝΓcDNA.

FIG. 3 is a photograph of two western blots comparing the expression levels of neublastin protein in untransfected H1B5 cells (lane 1) and in the H1B5 cell line stably transfected with neublastin cDNA (lane 2), where the protein was analyzed either using At -2 (left blot; panel A), or using At-1 antibodies specific for neublastin (right blot; panel B).

FIG. 4 is a graphic illustration of the effect of neublastin on the survival of rat embryo dopaminergic neurons cultured in serum-free dopaminergic neurons and on CAT activity in cholinergic cranial nerve motor neurons. In particular, figa is an illustration of the curve of the dependence of HAT activity on the dose of recombinant ΟΌΝΓ (dec. / Min / h). FIG. 4B is an illustration of HAT activity (dec. / Min / hr) when using a diluted conditioned medium from either neublastin-producing or ΟΌΝΓ-producing cells. FIG. 4C is an illustration of the number of immunoreactive cells per tyrosine hydroxylase assay.

FIG. Figure 5 illustrates the effect of neublastin secreted from ΗίΒ5ρυΜ1ζΝΒΝ22 cells on the functioning and survival of cultured sections of dopaminergic neurons of the ventral surface of the middle median bladder of pig embryos, cultivated together with either ΗίΒ5ρυΜ1ζΝΒΝ22 cells (neublastin) or with H1B5 cells (control). In FIG. 5A and FIG. 5B, dopamine is still released, released on Wednesday in I1U12 [dopamine (pmol / ml) -day 12] and I1U21 [dopamine (pmol / ml) -day 21], respectively. FIG. 5C is an illustration of the number of immunoreactive cells in a tyrosine hydroxylase assay in culture [TG-ir cells in culture] in I1U21.

FIG. 6 is an illustration of the effect of neublastin produced by lentiviruses on dopamine neurons of the black substance ίη νίνο.

FIG. 7 is a schematic diagram of the genomic structure of a neublastin gene, including nucleotide primers that can be used to identify a full-sized neublastin gene, and their spatial orientation with respect to the genomic sequence encoding neublastin (i.e., relative to the gene).

FIG. 8 is an illustration of neublastin-specific primers used to identify a cDNA clone encoding a human neublastin polypeptide that hybridize to nucleic acids that encode neublastin polypeptides but do not hybridize to nucleic acids encoding other known members of the ΟΌΝΓ family (i.e., ΟΌΝΓ perseffin and neurturin).

In FIG. Figure 9 shows the neurotrophic activity of the polypeptides of this invention in cultures of dissociated rat ganglion ganglion cells at different stages of development compared with activities achieved in the presence of known neurotrophic factors [0 control experiment (in the absence of factors)]; 1 in the presence of ΟΌΝΓ; 2 in the presence of neurturin; 3 in the presence of the neublastin of the invention; E12 - 12th embryonic day; E16 - 16th embryonic day; P0 - birthday; P7 - 7th day after birth; and P15-15th day after birth].

Figure 10 shows the production of neublastin with CHO cell lines.

In FIG. Figure 11 shows a comparison of the binding of neublastin and ΟΌΝΓ to the SRK-1 and SGK-3 receptors.

12 is a photograph of a Western blot showing the binding of the K.30 antipeptide antibody and K31 antipeptide antibody to neublastin.

13 is a gel image showing the extraction of neublastin by affinity binding to KET3-1d.

FIG. 14 is a map of the plasmid pET19b-neublastin along with the sequence of the synthetic gene of neublastin.

FIG. 15 is a map of the plasmid pM! B 164-H18-neublastin, along with the sequence of the synthetic Ηίκneiblastin gene.

DETAILED DESCRIPTION OF THE INVENTION

Applicants have identified a nucleic acid that encodes a new neurotrophic factor, which is here referred to as neublastin or ΝΒΝ. Neublastin is a member of the subclass of the neurotrophic factor obtained from the glial cell line (ΟΌΝΡ) of the superfamily of neurotrophic factors of transforming growth factor-β (ΤΟΡ-β).

cDNA encoding neublastin was originally identified as follows. First, using the algorithm ΤΒΕΑ8ΤΝ 1.4.11 (Αΐκοΐιι.11 е! А1., ΝποΙ.Αοίάκ Kek., 1997, 25, 33893402) and the person perseffin as a request (OepWapk Inv.No. ΑΡ040962), a fragment 290 bp long. in the high-performance genomic sequence (ΗΟΤ8) of two artificial bacterial chromosomes containing human DNA (IBC), was identified with OepBapk elements AC005038 and AC005051. AC005038 consists of approximately 190,000 bp 5 contigs of disordered sequences and AC005051 consists of approximately 132,000 bp 12 contigs of unordered sequences. It was proved that the 290 bp fragment identified in two IBC clones had regions that were homologous but not identical to the coding region of the human neurotrophic factor persefin cDNA.

On this sequence, 290 bp two PCR primers specific for neublastin were synthesized (upper chain primer | 8ES ΙΌ ΝΟ: 17] and lower chain primer | 8EC GO ΝΟ: 18]). A human cDNA library cDNA library was screened. Initial screening included a 96-well screening based on PCR with two PCR primers | 8ES GO 17: 17 and 18], a cDNA library in the main Mac! EG P1a! E plate with 500,000 clones of cDNA containing approximately 5000 clones / well. A second PCR-based test was performed on a human embryonic brain cDNA library in a 8iBP1a! Sub-plate containing a glycerol culture of approximately 25,000 clones / well.

A fragment of 102 bp ^ EC GO ΝΟ: 13] was identified by PCR-based screening of both libraries in the main plate (Mac! EG P1a! E) and the sub-plate (8uB P1a! E). A positive cDNA clone (containing a 102 bp fragment) was selected and plated on two plates containing LB / antibiotic and grown overnight. A total of 96 bacterial colonies were selected from these plates and separately plated in the wells of a new 96-well PCR plate containing both PCR primers Е EC GO ΝΟ: 17 and 18] and all necessary reagents for PCR amplification.

Then, PCR amplification was performed and 96 individual PCR reactions were analyzed by electrophoresis on a 2% agarose gel. Then, a positive colony with a clone containing a fragment of 102 bp was identified. Plasmid DNA was obtained and sequenced from a positive colony containing a 102 bp fragment. Subsequent analysis by sequencing revealed the presence of a full-sized cDNA of 861 bp ^ EC GO ΝΟ: 8]. 663 bp open reading frame (OPC) or coding region (CGO8). identical to 8EC GO ΝΟ: 8, encodes a pre-pro-polypeptide (named pre-pro-neublastin) and shown in 8EC GO ΝΟ: 9. Based on 8EC GO ΝΟ: 9, three variants of neublastin polypeptides were identified. These options include:

(ί) a polypeptide of 140 AK, referred to herein as 40140, which has the amino acid sequence designated 8EC GO ΝΟ: 10;

(ίί) a polypeptide of 116 AK, termed ΝΒΝ116 here, which has the amino acid sequence designated 8EC GO ΝΟ: 11; and (ίίί) a polypeptide of 113 AK, referred to herein as ΝΒΝ113, which has an amino acid sequence designated 8EC GO ΝΟ:

12.

The complete cDNA sequence containing 782 bp 5 'untranslated DNA, 663 bp coding DNA and 447 bp 3 'untranslated DNA (totaling 1992 bp) was presented in Geomna under accession number ΑΡ 120274.

The genomic sequence encoding neublastin was identified as follows.

In order to clone the genomic sequence encoding neublastin, an additional set of primers was obtained. In particular, the pair of primers No. 1 included [semantic = 8EC GO ΝΟ: 23 and the antisense = 8EC GO ΝΟ: 24] and the pair of primers No. 2 included [semantic = 8EC GO ΝΟ: 25 and antisense = 8EC GO ΝΟ: 26].

Using a pair of primers No. 2, a 887 bp DNA fragment was amplified from PCR from a human genomic DNA preparation, cloned into puigodep pC11 vector) and introduced into Ε.οοίί as a result of transformation. The resulting plasmid was sequenced, and the estimated cDNA sequence of 861 bp (coding for a protein called neublastin here) was calculated (as indicated in 8EC GO ГО: 3). Similarly, using a pair of primers No. 1 as a result of PCR of human genomic DNA, a DNA fragment of 870 bp was obtained. In this fragment, in comparison with a sequence of 887 bp in length, an additional region of 42 bp was found. at the 3'-end of the open reading frame (OPC). The genomic structure of the neublastin gene was predicted by comparing it with the nucleic acid sequences of other neurotrophic factors, by mapping exonintron boundaries. This analysis showed that the neublastin gene has at least two exons separated by an 70 bp intron.

This sequence has also been used to screen SeiBapk for the presence of E8T neublastin sequences. Three sequences were identified using SepBapk elements AA844072, AA931637 and AA533512, which indicates that neublastin nucleic acids are transcribed into mRNA.

Comparison of the complete sequence of the obtained cDNA (AG 120274) and the genomic sequence present in the SepBapk elements GS005038 and GS005051 showed that the neublastin gene consists of at least five exons (including three coding ones) separated by four introns (see, for example, Fig. 8). Together, exons have the predicted sequence for the amino acids of the full length neublastin polypeptide. It should also be noted that it was found that the fragment is 887 bp in length. contains the complete pro-neublastin coding region. The calculated cDNA | 8EC ΙΌ N0: 3] contains an open reading frame (OPC) encoding pro-neublastin (181 amino acid residues), which exhibits homology with three well-known human proteins - perseffin, neurturin and ΟΌΝΕ.

Neublastin Nucleic Acids of the Present Invention

Another aspect of the invention is the provision of polynucleotides capable of expressing the polypeptides of the invention. Polynucleotides of the invention include DNA, cDNA and RNA sequences, as well as antisense sequences, and include naturally occurring polynucleotides, synthetic and specially prepared polynucleotides. Polynucleotides of the invention also include sequences that are genetically degenerate but which encode the expression of a neublastin polypeptide.

As defined herein, the term polynucleotide refers to a polymeric form of nucleotides of at least 10 nucleotides in length, preferably at least 15 nucleotides in length. By an isolated polynucleotide is meant a polynucleotide that does not directly contact two coding sequences with which it is directly adjacent (one at the 5 'end and one at the 3' end) in the genome of natural origin of the organism from which it is derived. Therefore, the term includes recombinant DNA that is inserted into an expression vector, an autonomously replicating plasmid or virus, or a prokaryotic or eukaryotic genomic DNA, or that exists as a single molecule, such as cDNA, independently of other sequences.

The polynucleotides of the invention also include allelic variants and mutant polynucleotides having a nucleotide sequence that differs from the nucleotide sequence presented herein at one or more nucleotide positions.

In a preferred embodiment, the polynucleotide of the invention has a nucleic acid sequence (DNA) capable of hybridizing with a polynucleotide sequence represented by 8EC ΙΌ N0: 1, a polynucleotide sequence represented by 8EC ΙΌ N0: 3, a polynucleotide sequence represented by 8EC ΙΌ N0: 8, or a polynucleotide sequence represented as 8EC Е N0: 15, with strands complementary to them or their sub-sequences with at least medium, medium / hard or very sharp s stringent conditions as described below in more detail.

In another preferred embodiment, an isolated polynucleotide of the invention has a nucleic acid (DNA) sequence that is at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% homologous to the polynucleotide sequence presented in the form of 8EC ΙΌ N0: 1, the polynucleotide sequence represented in the form of 8EC пол N0: 3, the polynucleotide sequence represented in the form of 8EC ΙΌ N0: 8, or the polynucleotide Consistency, presented as 8EC ΙΌ N0: 15.

In a most preferred embodiment, the polynucleotide has a DNA sequence represented by 8EC ΙΌ N0: 1, a DNA sequence represented by 8EC ΙΌ N0: 3, a DNA sequence represented by 8EC ΙΌ N0: 8, or a polynucleotide sequence represented by 8EO ΙΌ N0: 15.

The invention also provides novel primers and DNA sequences for the identification, isolation and amplification of neublastin polynucleotides that encode the expression of neublastin polypeptides or fragments thereof. Such primers include polynucleotides shown in 8EC ΙΌ N0: 17-28 and 31-32. In addition, the present invention provides neublastin DNA sequences formed from these primers, including the sequences shown in 8E0 ΙΌ N0: 13 and 14. In addition, this invention provides DNA sequences from 3 'or 5' untranslated regions (UTR) of genomic DNA, which flank exons of neublastin; such sequences are useful for identifying, isolating, and amplifying neublastin polynucleotides that encode the expression of neublastin polypeptides or fragments thereof.

3'UTO sequences of the invention include sequences represented by the following nucleotides:

nucleotides 721-865 in 8E0 ΙΌ N0: 1, nucleotides 718-861 in 8E0 ΙΌ N0: 3, nucleotides 718-861 in 8E0 ΙΌ N0: 8, nucleotides 1647-2136 in 8E0 ΙΌ N0: 15 and adjacent sequences from 10 to 25 nucleotides derived from (i.e., being part of) the preceding sequences (which are suitable, for example, as primers).

5'UTO sequences of the invention include sequences represented by the following nucleotides:

nucleotides 1-10 in 8E0 ΙΌ N0: 1, nucleotides 1-57 in 8E0 ΙΌ N0: 8, nucleotides 1-974 in 8E0 ΙΌ N0: 15, and adjacent sequences from 10 to 25 nucleotides derived from (i.e., being part) of the preceding sequences (which are suitable, for example, as primers).

The polynucleotides of the invention can preferably be obtained using cloning procedures, for example, as described in Sitgos RtoFsok ίη Mo1eci1ag VF1od [Fip XVPSu & 8opk, FS.]. In a preferred embodiment, the polynucleotide is cloned from or prepared on the basis of human genomic DNA or a human brain cDNA library.

DNA sequence homology

The homology of the DNA sequences indicated above can be determined by the degree of identity between the two sequences, showing the origin of the first sequence from the second. Homology can be appropriately determined using computer programs known in the art, such as the OAR, provided in the CCA software package [No. 7, 8 .B. apy ΧνιιηκΟι S.E. 1oitpa1 ok Mo1esi1ag VF1od 1970, 48, 443-453]. Using the OAR with the following settings for comparing DNA sequences: a penalty score when creating a GAP 5.0 and a penalty score when expanding a GAP of 0.3, the coding regions of the above similar DNA sequences show an identity of preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, with the SI8 (coding) part of the DNA sequence shown in 8E0 ΙΌ N0: 1, or SI8 (coding) part followed the DNA sequence shown in 8E0 ΙΌ N0: 3, or SI 8 (coding) part of the DNA sequence shown in 8E0 ΙΌ N0: 8, CE8 (coding) part of the DNA sequence shown in 8EO ΙΌ N0: 15.

The term sequence identity refers to the degree to which two polynucleotide sequences are identical at the base of the nucleotide after the nucleotide throughout a particular comparison region. The term percent sequence identity refers to a percentage calculated by comparing two optimally aligned sequences across this comparison area, determining the number of positions at which identical nucleic acid bases (e.g. A, T, C, O, and or Ι) occur in both sequences to get the number of matching positions by dividing the number of matching positions by the total number of positions in the comparison area (i.e. window size) and multiplying the result by 100 to get the percent identity sequence identities. The term significant identity, as used herein, means a characterization of a polynucleotide sequence in which the polynucleotide includes a sequence that is at least 80% identical in sequence, preferably at least 85% identical, and often 90-95% identical in sequence, more often at least 99% identical in sequence to the reference sequence throughout the comparison area.

Hybridization protocol

Polynucleotides of the invention are those polynucleotides that have a nucleotide sequence capable of hybridization with the polynucleotide sequence represented in 8E0 ΙΌ N0: 1, with the polynucleotide sequence represented in 8E0 ΙΌ N0: 3, or with the polynucleotide sequence represented in 8E0 ΙΌ N0: 8 or with the polynucleotide sequence represented in 8E0 ΙΌ N0: 15, or with their complementary chains, or with their sub-sequences, at least with medium, medium / hard or very gesture conditions, as described in more detail below.

An acceptable experimental mode for determining hybridization between a nucleotide probe and a homologous DNA or RNA sequence includes pre-soaking a filter containing DNA or RNA fragments for hybridization at 5 x 88 ° C [sodium chloride / sodium citrate; compare

8th ego! A1 .; Mo1esi1ag S1oshpd: A aota OSU Mapia1, Co 16 8rtshd Nagog bab, Co 16 8rtshd Nagog, ΝΥ 1989] for 10 min, and prehybridization of the filter in a solution of 5 x 88c, 5 x Denhardt's solution [cf. 8attook e!.! A1 .; work already cited], 0.5% 8–8 and 100 µg / ml of denatured voiced salmon sperm DNA [cf. A1 .; work already cited] followed by hybridization in the same solution containing that obtained by the method of scattered seeding [ARET & R. Wodeschess .; ApaEBusbet., 1983, 132, 6-13], labeled with ³² R-dTCP (specific activity> 1 x 10 ⁹ cpm / μg) probe, at a concentration of 10 ng / ml, for 12 hours at a temperature of about 45 ° C . Then the filter was washed twice for 30 minutes in 0.1 x 88 ° C, 0.5% 8–8 at a temperature of at least 60 ° C (medium hardness conditions), preferably at least 65 ° C (medium / high stiffness), more preferably at least 70 ° C (high stringency conditions) and even more preferably at least 75 ° C (very high stringency conditions). Molecules with which the oligonucleotide probe hybridizes under these conditions can be detected using an X-ray film.

Cloned Polynucleotides

An isolated polynucleotide of the invention, in particular, may be a cloned polynucleotide. As defined here, the term cloned polynucleotide refers to a polynucleotide or DNA sequence cloned in accordance with standard cloning procedures currently used in genetic engineering to move a segment of DNA that, in particular, can be represented by cDNA, i.e. DNA obtained enzymatically from RNA from its natural position to another site where it will be reproduced.

Cloning can be carried out in various acceptable ways and can include techniques such as technology based on reverse transcriptase, PCR technology and the like, as well as cutting and isolation of the desired DNA segment.

The cloned polynucleotide of the invention may alternatively be called a DNA construct or an isolated DNA sequence, and may, in particular, be complementary DNA (cDNA).

Biological sources

An isolated polynucleotide of the invention may be obtained from any suitable source.

In a preferred embodiment, the polynucleotide of the invention is cloned or created on the basis of a cDNA library, for example, a cDNA library of a fetal or adult brain, in particular the forebrain, hindbrain, cerebral cortex, striatum, amygdala, cerebellum, caudate nucleus, corpus callosum, hippocampus , nuclei of the thalamus, nuclei of the hypothalamus, olfactory nucleus, shell, black matter, ganglion of the posterior root, trigeminal ganglion, nerve, superior mesenteric artery or thalamus; spinal cord; heart the placenta; lung; liver skeletal muscle; kidneys pancreas; intestines; eyes; retina tooth pulps; hair follicle; prostate gland; pituitary gland; or trachea.

Commercial cDNA libraries of various tissues, both human and other organisms, can be purchased, for example, in 81g1adepe and S1op1es11. An isolated polynucleotide of the invention can be obtained by standard methods, for example, methods described in the working examples.

Neublastin Polypeptides of the Present Invention

As noted above, the neublastin polypeptide in the sense used here is a polypeptide that has neurotrophic activity (for example, as described in examples 6, 7, 8 and 9) and this term includes those polypeptides that have an amino acid sequence that has, at least 70% homology with neublastin polypeptides represented by AK _-95- AK1 ₀₅ in 8EC Ц ΝΟ: 2, AK ₁ AK ₁₀₅ in 8ES GO ΝΟ: 2, AK _-97- AK ₁₄₀ in 8ES GO ΝΟ: 4, AK _-41 -AK ₁₄₀ to 8ES GO ΝΟ: 4, ASH-AK ^ to 8 EU) GO ΝΟ: 4, AK _-80 -AK ₁₄₀ to 8 EU) GO 9: 9 (wild type prepro), AK_ ₄ 1-AK1 ₄₀ 8ETS GO νΟ: 9 (pro), AA _£ -ak ₁₄₀ 8ETS GO ΝΟ: 5 (mature 140 AA), AA ₁ -ak ₁₁₆ 8 EC) GO ΝΟ: 6 (mature 116 AA), AA ₁ - ACC ₃ in the 8th CEC GO ΝΟ: 7 (mature, 113 AK), AK! -AK ₁₄₀ in the 8th EU) GO 10: 10 (mature, 140 AK), AK _G AK ₁₁₆ in 8 EU) GO ΝΟ: 11 (mature, 116 AK), AK _G AK ₁₁₃ in 8 EU) GO ΝΟ: 12 (mature, 113 AK), AA! -AA ₂₂₄ in 8 EU) GO ΝΟ: 16 (prepro mouse) and variants and derivatives of each polypeptide mentioned above.

Preferably, the C-terminal sequence of the neublastin polypeptides described above has the amino acid sequence represented by AK72-AK105 at 8EC) GO ΝΟ: 2 (i.e., AK ₁₀₇ -AK ₁₄₀ at 8ES) GO ΝΟ: 9), more preferably AK ₄₁ -AK ₁₀₅ in the 8th EC GO ΝΟ: 2 (i.e. AK ₇₆ -AK ₁₄₀ in 8ES) GO ΝΟ: 9).

It is also preferred that 7 conserved Cy8 residues that are characteristic of the семейства family and the TSE-beta superfamily are retained in the neublastin polypeptide.

Preferably, the neublastin polypeptide has an amino acid sequence more than 85% homologous, more preferably more than 95% homologous to the sequences mentioned above (i.e., AK _-95 -AK ₁₀₅ in 8EC) GO ΝΟ: 2, ASH-AK ^ in 8 EU) GO ΝΟ: 2, AK _-97 -AK ₁₄₀ to 8 EU) GO ΝΟ: 4, AK _-41 AK ₁₄₀ to 8ES) GO ΝΟ: 4, AKRAK ^ to 8ES) GO ΝΟ:

4, LC. ₈ o-LK ₁₄ o in 8EC ΙΌ N0: 9 (wild type prepro), AK _-41 -AK ₁₄₀ in 5EO ΙΌ N0: 9 (pro), AK ₁ AK ₁₄₀ in 8E0 ΙΌ N0: 5 (mature, 140 AK) , AK ₁ AK ₁₁₆ at 8E0 ΙΌ N0: 6 (mature, 116 AK), AK ₁ AK ₁₁₃ at 8E0 ΙΌ N0: 7 (mature, 113 AK), AK ₁ AK ₁₄₀ at 8E0 ΙΌ N0: 10 (mature, 140 AK ), AK ₁ AK ₁₁₆ at 5EO ΙΌ N0: 11 (mature, 116 AK), AK ₁ AK ₁₁₃ at 5EO ΙΌ N0: 12 (mature, 113 AK), AK ₁ AK ₂₂₄ at 8EC ΙΌ N0: 16 (prepro mouse) ), and preferably any of the aforementioned polypeptides with a C-terminal sequence of the neublastin polypeptides described above has the amino acid sequence represented by AK ₇₂ -AK ₁₀₅ in 8EC ΙΌ N0: 2 (i.e. AK ₁₀₇ -AK ₁₄₀ in 5EO Ι Ό N0: 9), more preferably AK ₄₁ -AK ₁₀₅ in 8EC ΙΌ N0: 2 (i.e. AK ₇₆ -AK ₁₄₀ in 5EO ΙΌ N0: 9) or AK ₁₉₁ AK ₂₂₄ in 5EO ΙΌ N0: 16.

In addition, the invention contemplates polypeptides that have an amino acid sequence that is at least 70% homologous to mouse neublastin polypeptides represented by AK ₁ -AK ₂₂₄ in 5EO О N0: 16.

Among the preferred polypeptides of the invention, in one embodiment, a prepro sequence (as presented in 8EC ΙΌ N0: 2, 4, 9 and 16, respectively), prosequence (as represented by AK- ₇₅ AK ₁₀₅ in 8EC ΙΌ N0: 2 or AK _-41 - AK ₁₄₀ in 8EC ΙΌ N0: 4 and 9, respectively) and the sequence of mature neublastin (as presented in 8EC ΙΌ N0: 5, 6, 7, 10, 11 or 12, preferably 8E0 ΙΌ N0: 10, 11, 12).

Polypeptides of the invention include variants of the polypeptides. In the context of this invention, the term polypeptide variant means a polypeptide (or protein) having an amino acid sequence that differs from the sequence represented by 8E0 ΙΌ N0: 2, 5EO ΙΌ N0: 4, 5EO ΙΌ N0: 5, 8E0 ΙΌ N0: 6, 5EO ΙΌ N0: 7, 5 EU) ΙΌ N0: 9, 5EC) ΙΌ N0: 10, 5EC) ΙΌ N0: 11, 5EC) ΙΌ N0: 12 or 8EC ΙΌ N0: 16 in one or more amino acid positions. Such polypeptide variants include the modified polypeptides described above, as well as conservative substitution polypeptides, splicing variants, isoforms, homologs of other species, and polymorphic variants.

As defined here, the term conservative substitutions means the replacement of an amino acid residue with another, biologically similar residue. For example, conservative amino acid substitutions should be expected to have little or no effect on biological activity, especially if they represent less than 10% of the total residues in the polypeptide or protein. Preferably, conservative amino acid substitutions are changes of less than 5% of the polypeptide or protein, most preferably less than 2% of the polypeptide or protein (for example, when the calculation was carried out with respect to IV # 13, most preferably conservative substitutions were represented by less than 3 amino acid substitutions in the mature amino acid wild type sequences). In a particularly preferred embodiment, there is a single amino acid substitution in a mature sequence where both amino acids, substituted and substituted, are non-cyclic.

Other specific examples of conservative substitutions include substituting one hydrophobic residue, such as isoleucine, valine, leucine or methionine, for another residue, or replacing one polar residue with another, such as replacing lysine with arginine, aspartic acid, glutamic acid or asparagine glutamine, and the like.

The term conservative substitution also includes the use of a substituted amino acid residue in place of an unsubstituted starting amino acid residue, provided that antibodies produced to the substituted polypeptide are also immunoreactive with respect to the unsubstituted polypeptide.

Modifications of this primary amino acid sequence can result in proteins that generally have equivalent activity compared to an unmodified copy of the polypeptide, and thus can be considered functional analogues of the original proteins. Such modifications may be intentional, for example, as modifications by site-specific mutagenesis, or they may occur spontaneously and include splicing variants, isoforms, homologues of other species, and polymorphic forms. Such functional analogs are also contemplated according to this invention.

In addition, modifications of the primary amino acid sequence can result in proteins that do not preserve the biological activity of the original protein, including dominant negative forms, etc. A dominant negative protein can compete with a wild-type protein by binding or otherwise by forming a complex with regulatory agents, such as upstream or downstream components, which usually functionally interact with the polypeptide. Such dominant negative forms are also contemplated according to the invention.

Signal peptide means a peptide sequence that directs the synthesized polypeptide with which this signal peptide is connected to the endoplasmic reticulum for further post-translational processing and placement.

A heterologous signal peptide, as used herein in the context of neublastin, means a signal peptide that is not a human neublastin signal peptide, typically a signal peptide of a mammalian protein other than neublastin.

One skilled in the art will recognize that a human neublastin DNA sequence (either cDNA or genomic DNA), or sequences that differ from human neublastin DNA, either by silent changes in codons or changes in codons that cause conservative amino acid substitutions, can be used to genetically modify cultured human cells, so that they will overexpress and secrete the enzyme.

The polypeptides of the present invention also include chimeric polypeptides or cleavable hybrid polypeptides in which another polypeptide fuses to the или-terminal or C-terminal of the polypeptide or fragment thereof. A chimeric polypeptide can be obtained by fusing a nucleic acid sequence (or part thereof) encoding another polypeptide with a nucleic acid sequence (or part thereof) of the present invention.

Methods for producing chimeric polypeptides are standard methods. Such methods usually require joining the sequences in such a way that they both end up in the same reading frame, and the expression of the hybrid polypeptide is controlled by the same promoter (s) and terminator.

The polypeptides of this invention also include truncated forms of a full-sized neublastin molecule. In such truncated molecules, one or more amino acids are deleted at the Ν end or at the C end, preferably at the конце end.

Homology of amino acid sequences

The degree of homology of the polypeptide selected for testing with the neublastin polypeptide of the invention is defined as the degree of identity between two amino acid sequences. A high level of sequence identity indicates the likelihood that the first sequence is derived from the second.

Homology is determined by computer analysis; this is, but is not limited to, analysis by means of a C1k1a1X computer alignment program [Tkotkop GO, C1kkop T1, P1e \\ 'shak P, 1eapoidt R. & Ηί§shpk ΌΟ: Tke C1k1a1X \\' k16o || ШсШс Ααάκ Век., 1997, 25 (24): 4876-82], and the default parameters are proposed here. Using this program, it was shown that the mature portion of the polypeptide encoded by a similar DNA sequence of the invention exhibits a degree of identity of at least 90%, more preferably at least 95%, most preferably at least 98%, s the amino acid sequence presented here in the form of BEC GO N0: 2, BEC GO N0: 4, BEC GO N0: 5, BEC GO N0: 6, BEC GO N0: 7, BEC GO N0: 9, BEC GO N0: 10, BEC GO N0: 11, WEATHER GO N0: 12 or WEATHER GO N0: 16.

Based on the definition of homology, it was confirmed that the polypeptide of the invention related to the superfamily of TCR-β belongs to the subfamily of the secondary school, but is a special member of this family.

Biologically active polypeptides

The polypeptide of the invention may be in any biologically active form, including the form of pre-pro-proteins, whitespace, mature proteins, glycosylated proteins, phosphorylated proteins or any other post-translationally modified protein.

The polypeptide of the invention, in particular, can be a glycosylated polypeptide, and this polypeptide is preferably glycosylated at the N-residues indicated in the sequence lists.

In a preferred embodiment, the polypeptide of the invention has an amino acid sequence presented as BECHO GO N0: 9, containing a glycosylated asparagine residue at position 122; the amino acid sequence presented in the form of BES GO N0: 10 containing a glycosylated asparagine residue at position 122; the amino acid sequence presented in the form of BES GO N0: 11 containing a glycosylated asparagine residue at position 98; or the amino acid sequence presented in the form of BES GO N0: 12 containing the glycosylated residue of asparagine at position 95.

The invention also contemplates neublastin fusion proteins, such as 1d hybrids, as described, for example, in US Pat. No. 5,434,131, incorporated herein by reference.

In one embodiment, the invention provides a polypeptide having the amino acid sequence shown as BECHO GO N0: 2, or an amino acid sequence that is at least about 85%, preferably at least about 90%, more preferably, at least about 98% and most preferably at least about 99% homologous to the sequence represented as BECHO GO N0: 2.

In another embodiment, the invention provides a polypeptide having an amino acid sequence of BECHO GO N0: 4 or an amino acid sequence that is at least 90%, more preferably at least 95%, even more preferably at least 98 %, most preferably at least 99% homologous to the sequence presented in the form of 8EO GO N0: 4.

In a third embodiment, the invention provides a polypeptide having an amino acid sequence of 8E0 GO N0: 5 or an amino acid sequence that is at least 90%, more preferably at least 95%, most preferably at least 98% homologous to the sequence represented by 8EO GO N0: 5.

In a fourth embodiment, the invention provides polypeptides having an amino acid sequence of 8E0 GO N0: 6 or an amino acid sequence that is at least 90%, more preferably at least 95%, most preferably at least 98% homologous to the sequence represented as 8E0 GO N0: 6.

In a fifth embodiment, the invention provides polypeptides having an amino acid sequence of 8E0 GO N0: 7 or an amino acid sequence that is at least 90%, more preferably at least 95%, most preferably at least 98% homologous to the sequence represented by 8EE) GO N0: 7.

The neublastin polypeptide of the invention includes allelic variants, for example, amino acid sequences of 8E0 GO N0: 5-7 polypeptides in which Xaa is Aki or T11G. and Waa means A1a or Prgo.

In a sixth embodiment, the invention provides polypeptides having an amino acid sequence of 8E0 GO N0: 9 or an amino acid sequence that is at least 90%, more preferably at least 95%, most preferably at least 98% homologous to the sequence represented as 8E0 GO N0: 9.

In a seventh embodiment, the invention provides a polypeptide having an amino acid sequence of 8E0 GO N0: 10 or an amino acid sequence that is at least 90%, more preferably at least 95%, most preferably at least 98% homologous to the sequence represented by 8EE) GO N0: 10.

In an eighth embodiment, the invention provides a polypeptide having an amino acid sequence of 8E0 GO N0: 11 or an amino acid sequence that is at least 90%, more preferably at least 95%, most preferably at least 98 % homologous to the sequence represented by 8EE) GO N0: 11.

In a ninth embodiment, the invention provides a polypeptide having an amino acid sequence of 8E0 GO N0: 12 or an amino acid sequence that is at least 90%, more preferably at least 95%, most preferably at least 98% homologous to the sequence represented by 8EE) GO N0: 12.

In a tenth embodiment, the invention provides a polypeptide having an amino acid sequence of 8E0 GO N0: 16 or an amino acid sequence that is at least 90%, more preferably at least 95%, most preferably at least 98% homologous to the sequence presented in the form of 8E0 GO N0: 16, which is pre-proneublastin, the source of which are mice.

In another embodiment, the polypeptide of the invention has a fingerprint of an SBDE subfamily, i.e. amino acid residues underlined in table 3.

In a further embodiment, the invention provides a polypeptide encoded by a polynucleotide sequence capable of hybridizing under high stringency conditions with a polynucleotide sequence represented as 8E0 GO N0: 1, with a chain complementary to it, or a sub-sequence thereof. In a preferred embodiment, the polypeptide of the invention is encoded by a polynucleotide sequence of at least 70% of the homologous polynucleotide sequence represented as 8E0 GO N0: 1. In a most preferred embodiment, the polypeptide of the invention is encoded by a polynucleotide sequence represented by 8E0 GO N0: 1.

In yet another embodiment, the invention provides novel polypeptides that are encoded by a polynucleotide sequence capable of hybridizing under high stringency conditions with a polynucleotide sequence represented as 8E0 GO N0: 3, with a complementary strand thereof or its subsequence. In a preferred embodiment, the polypeptide of the invention is encoded by a polynucleotide sequence of at least 70% of the homologous polynucleotide sequence represented as 8E0 GO N0: 3. In a most preferred embodiment, the polypeptide of the invention is encoded by a polynucleotide sequence represented by 8E0 GO N0: 3.

In yet another embodiment, the invention provides novel polypeptides that are encoded by a polynucleotide sequence capable of hybridizing under high stringency conditions with the polynucleotide sequence represented by ZEC GO N0: 8, with a chain complementary to it, or a subsequence thereof. In a preferred embodiment, the polypeptide of the invention is encoded by a polynucleotide sequence of at least 70% of the homologous polynucleotide sequence represented by ZEC GO N0: 8. In the most preferred embodiment, the polypeptide of the invention is encoded by a polynucleotide sequence represented by 8EC GO N0: 8.

In yet another embodiment, the invention provides novel polypeptides that are encoded by a polynucleotide sequence capable of hybridizing under high stringency conditions with a polynucleotide sequence represented by ZEC GO N0: 15, with a chain complementary to it or its subsequence. In a preferred embodiment, the polypeptide of the invention is encoded by a polynucleotide sequence of at least 70% of the homologous polynucleotide sequence represented by ZEC GO N0: 15. In a most preferred embodiment, the polypeptide of the invention is encoded by a polynucleotide sequence represented by 8EC GO N0: 15.

Biological source

The polypeptide of the invention can be isolated from mammalian cells, preferably from human cells or from mouse cells.

In a most preferred embodiment, the polypeptide of the invention can be isolated from human heart tissue, from human skeletal muscle, from the human pancreas, or from human brain tissue, in particular from the caudate nucleus or from the thalamus, or it can be obtained from DNA originating from cells mammals, as discussed in more detail below.

Neurotrophic activity

The neublastin polypeptides of the invention are useful for slowing down the metabolism, growth, differentiation or survival of nerves or nerve cells. In particular, neublastin polypeptides are useful for treating or alleviating a disorder or disease in existing animals, for example in humans, such a disorder or disease that is amenable to neurotrophic agents. Such treatments and methods of treatment are described in more detail below.

Antibodies

Neublastin polypeptides or polypeptide fragments of the invention are used to produce antibodies specific for neublastin. As used herein, a neublastin-specific antibody is an antibody, for example a polyclonal antibody or a monoclonal antibody, that is immunoreactive with a neublastin polypeptide or polypeptide fragment or that specifically binds to epitopes of neublastin polypeptides.

The preparation of polyclonal and monoclonal antibodies is well known in the art. Polyclonal antibodies can in particular be prepared as described, for example, Sgr. E! a1.,: Przobisyoi οί Po1us1op1 Aiyzeg in 1typos11st1sa1 Prgo! oso1z (Maizoi, Eb.); Nitaia Rgesz, 1992, Radez 1-5; COME E! A1.,: Prisoners и о 1 ус ус 1 1 1 1 А ий з ега ш з з,, К. К., К. К. з, М, M1ce aib Natz1egz in Siggei! Rgo1oso1z sh 1ttio1odu, 1992, 8soy 2.4.1; and Eb Naglote aib Eau1b baie (Ebz.) in Aiuobi ez: Aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa. Rgesz, 1988. These protocols are hereby incorporated by reference. Monoclonal antibodies can be, in particular, prepared as described, for example, Kojeg & Mbzet, No. 1g. 1975, 256: 495; Come out e! A1., in Siggei! Rgo1oso1z sh 1ttio1odu, 1992, 8soy 2.5.1-2.6.7; and Nag1ote e! A1., in Aiobi1ez: Aaaoga! ogu Maiaia1; Co1b 8rgd Nagbog, Puib., 1988, rad. 726; and these protocols are incorporated herein by reference.

Briefly, monoclonal antibodies can be obtained by injecting, for example, mice with a composition containing the antigen, checking for antibody production by taking a serum sample, extracting the spleen to obtain B lymphocytes, fusion of Wlimphocytes with myeloma cells to produce hybridomas, cloning hybridomas, selecting positive clones that produce antibodies against the antigen; and the isolation of antibodies from hybridoma cultures.

Monoclonal antibodies can be isolated and purified from hybridoma cultures using a variety of well-developed methods, including protein-A-Sepharose affinity chromatography, molecular size exclusion chromatography, and ion-exchange chromatography, see, for example, Come down! a1. in Siggei! Рго! ОСО1з ш 1ттиио1оду, 1992, 8soyoi 2.7.1-2.7.12, aib 8soyoi 2.9.1-2.9.3; and wagiez e! A1: Ripysayoi ί 1ίттииодододЬ С С С С 1 1 1д 1 1 в 1 1 1 1С 1) в в в в в в в в в М в в в в в вз ш в з ш ш Мо 11 1ю;;;; Nitaia Rgesz, 1992, Wo1.10, Radez 79-104. Polyclonal or monoclonal antibodies can then be optionally purified, for example, by binding and eluting from the matrix to which the polypeptide to which antibodies are generated is bound.

Antibodies that bind to the neublastin polypeptide of the invention can be prepared using the intact polypeptide of interest or fragments containing small peptides as the immunizing antigen. The polypeptide used to immunize an animal can be obtained using recombinant DNA technology or by chemical synthesis and can optionally be conjugated to a protein carrier. Commonly used protein carriers that chemically bind to the peptide include the clam hemocyanin saucer (keu1o1e Ntre!) (GMB), thyroglobulin, bovine serum albumin (BSA) and tetanus toxoid. The bound peptide can then be used to immunize an animal, which may be, in particular, a mouse, rat, hamster or rabbit.

In one embodiment, antibodies are prepared using the following peptides: peptide 1: ΟΒΡΤΚΎΕΑνδΡΜΌνΝδΤ (amino acids 108124 8EO GO N0: 9); or peptide 2:

AEIRRRS, 8IR \ 8ORS (amino acids 93-107 8E0 GO N0: 9). Methods for producing antibodies using these polypeptides are described in Example 10.

The authors also obtained rabbit polyclonal antibodies to the following peptides:

peptide K27: 6P08CAK.AA6AK.0C (amino acids 30-43 8E0 GO N0: 9);

K.28 peptide: L0HK8PEI \ CRKRS (amino acids 57-70 8E0 GO N0: 9);

K29 peptide: SCKAK.8PHNb8 (amino acids 74-85 8EO GO N0: 9);

peptide K30: RKRRRS18IR \ 8ORS (amino acids 94-107 8E0 GO N0: 9); and peptide K31: §T ^ CT \ 0KB8ATAC (amino acids 123-136 8EO GO N0: 9).

In this group, only antibodies against peptides K30 and K31 located relatively close to the C-terminus recognized the denatured protein under reducing conditions on a Western blot.

The authors also identified additional peptides derived from neublastin, obtained from a mature protein, as described in more detail below, which are putative loops exposed on the surface, this assumption was made on the basis of the well-known structure of the CEF (E1debg! Apb OeGe, No.! .8! !. Вю1., 1997, 4, 435-438), and therefore they are suitable for the formation of antibodies:

District 1: СРРР80Р \ Т \ ИАРС1РС111Р81) ЕР \ РРРРС (AK 43-70 8EO GO N0: 9);

District 2: SKK.AK.8RNP8BA8B6A6AB KRRRS18IR \ 8ORS (AK 74-107 8EO GO N0: 9);

District 3: SIRTI ¥ EA \ 8P \ 11) \ A8T \\ 'IT \ EX8ATAS (AK 108-136 8EO GO N0: 9).

According to another aspect of the invention, antibodies that specifically bind neublastin, or peptides derived from neublastin, can be used to detect the presence of such neublastin neurotrophic factors in various media, and in particular to diagnose conditions or diseases associated with the neublastin molecules of the invention. Numerous protocols for this identification are known in the art, including E8A, RIA and PAC8.

Antibodies of the invention can also be used to block the action of a neurotrophic factor, and, in particular, can be neutralizing antibodies.

Methods for producing polypeptides of the invention

A cell containing the DNA sequence encoding the neublastin polypeptide of the invention is cultured under conditions ensuring the production of the polypeptide, followed by extraction of the polypeptide from the culture medium, as described in more detail below. In the case when the cells must be genetically modified in order to obtain the neublastin polypeptide, the cells can be modified by conventional methods or by activating the gene.

According to traditional methods, a DNA molecule that contains a cDNA or genomic DNA sequence of neublastin can be in an expression construct and can be transfected into cells by standard methods, including, but not limited to, transfection mediated by liposomes, polybrene or DEAE dextran, electroporation, precipitation with calcium phosphate, microinjection, or high-speed guided microprojectiles (LSD). Alternatively, a system that delivers DNA using a viral vector can be used. Known viruses suitable for gene transfer include adenoviruses, adeno-associated virus, lentivirus, herpes virus, mumps virus, poliovirus, retroviruses, Sindbis virus and vaccinia virus, such as canary pox virus, as well as baculovirus infection of insect cells, in particular insect cells 8GR9.

Alternatively, the cells can be modified using a gene activation method (GA), such as described in US Pat. Nos. 5,733,761 and 5,750,376, each of which is incorporated herein by reference.

Thus, the term genetically modified, as used here in relation to cells, means the production of cells that express a particular gene product due to the introduction of a DNA molecule encoding a given gene product and / or regulatory elements that control the expression of a sequence encoding a gene product. A DNA molecule can be introduced by directing a gene to a specific target, which allows you to include a DNA molecule in a specific genomic site.

Recombinant Expression Vectors

According to a further aspect, the invention provides a recombinant expression vector comprising a polynucleotide of the invention. The recombinant expression vector of the invention may be any suitable eukaryotic expression vector. Preferred recombinant expression vectors are the pT1-8 vector containing the ubiquitin promoter PbcpcTe, 8c1eu11eg M8, Tok! Ou 8, 8c1taP / T; REV8LeR (1990, 276, 289-294), and its derivatives, for example ρυόίΙΖ. A preferred available commercial eukaryotic expression vector is, for example, the ροΟΝΑ-3 vector containing the viral promoter (available for purchase at 1pu | 1godep). In another preferred expression vector, the early promoter 8U40 and the main late promoter of adenovirus are used (obtained from plasmid pAE2beta; No. r1op apb Co1yp, Mo1.Ce11.Vy1., 1985, 5, 281).

The invention also provides prokaryotic expression vectors and synthetic genes (syngens) with codon optimization for expression in prokaryotes. Syngens with a lower OS content and preferably bacterial (e.g., E.coI) codons were constructed. Syngen was cloned in two vectors, pET19b and pM1B164, a derivative of pET19b. Structures with pET19 are shown in FIG. In this construct, the sequence encoding the mature neublastin domain is directly fused to the methionine initiation codon. A design with pM1B164 is shown in FIG.

Producer cells

According to another aspect, the invention provides producer cells which, as a result of genetic manipulations, comprise an isolated polynucleotide sequence of the invention and / or a recombinant expression vector of the invention. A cell of the invention can be, in particular, a cell obtained as a result of genetic manipulations that temporarily or stably expresses, overexpresses or co-expresses a polypeptide of the invention. Methods for producing transient or stable expression are known in the art.

A polynucleotide of the invention can be inserted into an expression vector, for example, a plasmid, virus or other expression carrier, and by ligation it is operably linked to expression control sequences so that expression of the coding sequence is achieved under conditions that combine with expression control sequences. Suitable expression control sequences include promoters, enhancers, transcription terminators, start codons, intron splicing signals, and stop codons, all of which are contained in the correct reading frame of the polynucleotide of the invention so as to ensure proper translation of the mRNA. Expression control sequences may also include additional components, such as leader sequences and hybrid partner sequences.

The promoter may be, in particular, a constitutive or inducible promoter. When cloning in bacterial systems, inducible promoters such as pb of bacteriophage λ, p1ac, p1gr, p1ac (p! Gr-1ac hybrid promoter) can be used. When cloning in mammalian systems, promoters derived from the mammalian cell genome, for example, the ubiquitin promoter, the TK promoter or the metallothionein promoter, or from mammalian viruses, for example, the long terminal repeat of retroviruses, the late adenovirus promoter, or the 7.5K vaccinia virus promoter, can be used. To ensure transcription of the polynucleotide of the invention, promoters obtained using recombinant DNA techniques or synthetically can also be used.

Typically, suitable expression vectors contain an expression start point, a promoter, as well as specific genes that allow phenotypic selection of transformed cells, and include vectors similar to the T7-based expression vector for expression in bacterial cells [Ref. A1 .; Sepe. 1987, 56, 125], pT1-8, p1b1, pcPt-3 and p1X1) expressing vectors for expression in mammalian cells [Bey apb No. 1yap8, 1. Vy1. Syet., 1988, 263, 3521], vectors derived from baculoviruses for expression in insect cells, and an oocyte expression vector Бог | Bogepkh S, Riksi M. & lep (5c11 T. 1: Not! νί11ι poue1 regoreyek; Proc. No. 111. Asab. 8cE and 8A 1996, 93, 13362-13366].

In a preferred embodiment, the cell of the invention is a eukaryotic cell, for example a mammalian cell, for example a human cell, an oocyte or a yeast cell. A cell of the invention, without limitation, may be a human embryonic kidney cell (HEK), for example, a HEK293 cell, a BHK21 cell, a Chinese hamster ovary cell (CHO), an oocyte cell Heporik 1aeg15 (XO). In another embodiment, the cell of the invention is a fungal cell, for example a filamentous fungal cell. In another preferred embodiment, the cell is an insect cell, most preferably cell 819. Additional preferred mammalian cells of the invention are PC12, H1B5, K. # 33b cell lines and human nerve cell precursor cells. Most preferred are human cells.

Examples of primary or secondary cells include fibroblasts, epithelial cells, including breast and intestinal epithelial cells, endothelial cells, blood cells, including lymphocytes, and bone marrow cells, glial cells, hepatocytes, keratinocytes, muscle cells, nerve cells, or precursors of these cell types. Examples of immortalized human cell lines suitable for use in the present methods include, but are not limited to, Bo \\ 'C5 melanoma cells (ATCC inventory Νο. CKB 9607), Όαιιάί cells (ATCC inventory Νο. CCB 213), cells Heb and derivatives of Heb cells (ATCC inventory Νοκ. CCB 2, CCB 2.1 and CCB 2.2), Hb-60 (ATCC inventory Νο. CCB 240), HT-1080 cells (ATCC inventory Νο. CCB 121), cells 1 thread1 (ATCC inventory Тο. T1B 152), KV carcinoma cells (ATCC inventory Νο. SSB 17), K-562 leukemia cells (ATCC inventory KSS 243), cancer cells can full-time gland MCP-7 (ATCC inventory .ο. BTN 22), MOT-4 cells (ATCC inventory Νο. 1582), ΝαιηαΡνα cells (ATCC inventory Νο. CKB 1432), Katz cells (ATCC inventory Νο. CCB 86), CRM1 cells 8226 (ATCC inventory .ο. SSB 155), I937 cells (ATCC inventory Νο. CKB 1593), subline U1-38UA13 cells 2K4 (ATCC inventory Νο. СЬ 75.1) and ovarian carcinoma cells 2780ΑΌ (Ub Bet Внек е! А1., Сап. Kek., 1988, 48, 5927-5932), as well as heterohybrid cells obtained by fusion of human cells and cells of other species. Secondary human fibroblast lines can also be used, such as 1-38 (ATCC inventory .ο. SSB 75) and MKS-5 (ATCC inventory Νο. SSB 171).

In the case where the cell of the invention is a eukaryotic cell, the inclusion of a heterologous polynucleotide of the invention can, in particular, be carried out by infection (using a viral vector), by transfection (using a plasmid vector), by precipitation with calcium phosphate, microinjection, electroporation lipofection or other physicochemical methods known in the art.

In a more preferred embodiment, the isolated polynucleotide sequence of the invention and / or the recombinant expression vector of the invention is transfected into a mammalian host cell, nerve cell progenitor cell, astroglial cell, T cell, hematopoietic stem cell, non-dividing cell or brain endothelial cell containing at least one DNA molecule capable of mediating cell immortalization and / or transformation.

Activation of the endogenous gene in the host cell can be accomplished by introducing regulatory elements, in particular by introducing a promoter capable of influencing the transcription of the endogenous gene encoding the neublastin polypeptide of the invention.

Pharmaceutical Compositions

According to another aspect, the invention provides novel pharmaceutical compositions comprising a therapeutically effective amount of a polypeptide of the invention.

For therapeutic use, the polypeptide of the invention may be introduced in any suitable form. In a preferred embodiment, the polypeptide of the invention is included in the pharmaceutical composition together with one or more adjuvants, excipients, carriers and / or diluents, and the pharmaceutical composition is prepared by a person skilled in the art using standard methods known in the art.

Such pharmaceutical compositions may contain a polypeptide of the invention or an antibody against it. The composition may be administered alone or in combination with one or with a large number of other agents, drugs or hormones.

The pharmaceutical composition of the present invention can be administered by any suitable method, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intra tracheal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, small intestine, local, sublingual or , buccal, vaginal, intraocular, intracerebral, intracranial, intraspinal, intraventricular, intracistern, intracapsular nd, pulmonary, mucosally, or by inhalation.

Methods, apparatus, and preparation of a medicament for intrapulmonary delivery are described, for example, in US Pat. Nos. 5,785,049, 5,780,019 and 5,775,321, each of which is incorporated herein by reference. The administration can be in the form of periodic injections of shock doses of the drug, or it can be carried out more continuously by intravenous or intraperitoneal administration from a reservoir that is located outside (for example, balloon IV) or inside (for example, a biodegradable implant, an artificial biological organ, or a colony of implanted cells, producing neublastin). See, for example, US Pat. Nos. 4,407,957, 5,798,113 and 5,800,828, each of which is incorporated herein by reference. Methods and apparatus for intrapulmonary delivery are described, for example, in US patents 5654007, 5780014 and 5814607, each of which is incorporated herein by reference.

The administration of the neublastin according to this invention can be achieved, in particular, by any suitable delivery methods, including:

(a) a pump (see, for example, Appa1k ίί Ryagtasoegaru, 27: 912 (1993); Sapseg, 41: 1270 (1993); Sapseg Kekeagsy, 44: 1698 (1984), incorporated herein by reference), (b) microencapsulation (see, for example, US Pat. Nos. 4,352,883; 4,353,888 and 5,084,450, incorporated herein by reference), (c) a continuous release polymer implant (see, e.g., 8aBe1, US Pat. No. 4,883,666, incorporated herein by reference), (i) microencapsulation (see, for example, US patents 5284761, 5158881, 4976859 and 4968733 and published PCT patent applications \ U0 92/19195, \ U0 95/05452, each of which is incorporated here by reference), (e) uncovered or unencapsulated cell transplants in the central nervous system (see, for example, US Pat. Nos. 5,082,670 and 5,618,531, each of which is incorporated herein by reference), (ί) injections, or subcutaneous, intravenous, intraarterial, intramuscular, or another suitable place ; and (e) oral administration in a capsule, liquid, tablet, pill or sustained release composition.

In one embodiment of the present invention, neublastin is delivered directly to the central nervous system, preferably to the ventricles of the brain, brain parenchyma, subshell space, or to other suitable locations of the central nervous system, most preferably to the subshell space.

Another preferred option, the authors consider systemic delivery by subcutaneous injection, intravenous administration or intravenous infusion.

Other parenteral delivery systems used include ethylene vinyl acetate copolymer particles, an osmotic pump, implantable infusion systems, pump delivery, encapsulated cell delivery, liposome delivery, needle injection, needleless injection, nebulizer, aerosol mash, electroporation and transdermal patch.

Further details of the methods for preparing the composition and administration can be found in the latest edition of YttddUp'k Ryagtaseijs1 8s1epsek (Maask RyykYpd So., EakUp, RA).

The active ingredient may be administered in one or more doses per day. Dosages between 0.5 ng neublastin / kg body weight up to about 50 mcg / kg per administration and from about 1.0 ng / kg to about 100 mcg / kg per day are currently considered suitable. A pharmaceutical composition containing neublastin should provide a local concentration of neurotrophic factor from about 5 ng / ml cerebrospinal fluid (CSF) to 25 ng / ml CSF.

Of course, the administered dose should be carefully selected in accordance with the age, weight and condition of the person who is being treated, and also, of course, the practitioner must determine the route of administration, dosage form and dosage regimen, the desired result and the exact dose.

In a further embodiment, the neublastin polypeptide of the invention can be introduced using gene delivery, using cell lines and vectors, as described below in the treatment methods. To create such therapeutic cell lines, the polynucleotide of the invention can be inserted into an expression vector, for example, a plasmid, virus or other expression carrier, and linked to the same expression control sequences by ligation so that expression of the coding sequence is achieved under conditions compatible with expression control sequences. Suitable expression control sequences include promoters, enhancers, transcription terminators, start codons, intron splicing signals, and stop codons, all of which are contained in the correct reading frame of the polynucleotide of the invention so as to ensure proper translation of the mRNA. Expression control sequences may also include additional components, such as leader sequences and hybrid partner sequences.

The promoter may be, in particular, a constitutive or inducible promoter. Constitutive promoters can be synthetic, viral, or derived from the mammalian cell genome, for example, the human ubiquitin promoter. In a preferred embodiment, the therapeutic cell line will be a continuously dividing nerve cell line expressing a polypeptide of the invention. Upon implantation, the authors suggest implanting from about 10 ⁵ to 10 ¹⁰ cells, more preferably from 10 ⁶ to about 10 ⁸ cells.

Treatment methods

This invention, which relates to polynucleotides and proteins, polypeptides, peptide fragments or derivatives derived therefrom, as well as antibodies directed against such proteins, peptides or derivatives, can be used to treat or alleviate a disorder or disease in animals, including humans, those disorders or diseases that are susceptible to neurotrophic agents.

The polypeptides of this invention can be used directly, for example, by injectable, implantable or oral pharmaceutical compositions for the treatment of a pathological process sensitive to neublastin polypeptides.

Polynucleotides of the invention, including sequences complementary to them, can be used to express a neurotrophic factor of the invention. This can be achieved using cell lines expressing such proteins, peptides or derivatives of the invention, or using viral vectors encoding such proteins, peptides or derivatives of the invention, or using host cells expressing such proteins, peptides or derivatives. These cells, vectors and compositions can be introduced to treat the target region to affect the disease process sensitive to neublastin polypeptides.

Suitable expression vectors can be derived from lentiviruses, retroviruses, adenoviruses, herpes viruses, or smallpox viruses, or from plasmids derived from bacteria, and can be used to deliver nucleotide sequences to an entire organism or target as an organ, tissue or population cells. Other methods include, but are not limited to, liposome transfection, electroporation, carrier peptide transfection containing nuclear or other localization signals, and gene delivery via sustained release systems. According to another aspect of the invention, antisense nucleotide sequences complementary to the neublastin gene or parts thereof can be used to inhibit or enhance the expression of neublastin.

According to another aspect, the invention relates to a method for treating or alleviating a disorder or disease in animals, including humans, such a disorder or disease that is sensitive to the action of neurotrophic agents.

The disorder or disease can be, in particular, damage to the nervous system as a result of trauma, surgery, ischemia, infection, metabolic diseases, nutritional deficiencies, malignant processes or toxic agents, and genetic or idiopathic processes.

Damage can, in particular, occur in sensitive neurons or cells of the retinal ganglion, including neuron ganglion neurons or in any of the following tissues: knee ganglion, lower glossopharyngeal nerve ganglion or nodular ganglion; the vestibular-auditory complex of the vulvar cranial nerve; ventrolateral pole of the maxillary and mandibular lobe of the trigeminal nerve node; and the nucleus of the midbrain tract of the trigeminal nerve.

In a preferred embodiment of the method of the invention, the disease or disorder is a neurodegenerative disease in which damaged or injured neurons are involved, in particular traumatic injuries of peripheral nerves, the medulla oblongata and / or spinal cord, damage to neurons due to cerebral ischemia, neuropathy, and especially peripheral neuropathy, trauma or damage to the peripheral nerve, ischemic stroke, acute damage to the brain, acute damage to the spinal cord, about nervous system tumors, multiple sclerosis, exposure to neurotoxins, metabolic diseases such as diabetes mellitus or impaired renal function, and damage caused by infectious agents, neurodegenerative diseases, including Alzheimer's disease, Huntington's disease, Parkinson's disease, progressive Parkinson's disease, progressive supranuclear palsy (Steele-Richadson-Olszewski syndrome), olivopontotserebellar degeneration (SPDC), Shay-Draeger syndrome (multiple systemic atrophy), dem complex ntsiya-parkinsonism (Guam-type), amyotrophic lateral sclerosis, or any other congenital or neurodegenerative disease, and memory impairment associated with dementia.

The authors consider treatment of sensitive neurons and / or neurons of autonomous systems to be the preferred option. Another preferred option, the authors consider the treatment of diseases of motor neurons, such as amyotrophic lateral sclerosis (ALS) and spinal amyotrophy. Another preferred option, the authors consider the use of the neublastin molecules of the present invention to accelerate the recovery of nerves after a traumatic lesion. In one embodiment, the authors suggest the use of a nerve control channel using a matrix containing neublastin polypeptides. Such nerve control channels are claimed, for example, in US Pat. No. 5,843,029, incorporated herein by reference.

In a preferred embodiment, the polypeptides and nucleic acids of the invention (and pharmaceutical compositions that contain them) are used to treat peripheral neuropathies. Among peripheral neuropathies, it is contemplated to treat with the molecules of the invention trauma-induced neuropathies, such as those caused by physical damage or a pathological condition, physical damage to the brain, physical damage to the spinal cord, stroke related to brain damage, and neurological disorders associated with neurodegeneration.

The authors also suggest the treatment of chemotherapy-induced neuropathies (such as neuropathy caused by chemotherapeutic agents such as taxol or cisplatin); toxin-induced neuropathies, drug-induced neuropathies, vitamin-deficient neuropathies; idiopathic neuropathies; and diabetic neuropathies. See, for example, US patents 5496804 and 5916555, each of which is incorporated here by reference.

The authors also suggest the treatment of non-neuropathic diseases, multiple mononeuropathies, polyneuropathies, including axonal and demyelinating neuropathy, using nucleotides and polypeptides of the neublastin of the invention.

In another preferred embodiment, the polypeptides and nucleic acids of the present invention (and pharmaceutical compositions that contain them) are used to treat various eye diseases, including the destruction of retinal photoreceptors in patients with macular degeneration, retinitis pigmentosa, glaucoma and similar diseases.

Another object of the present invention is to provide a method for preventing degenerative changes associated with the above diseases and disorders by implantation in the brain of mammals, including humans, vectors or cells capable of producing a biologically active form of neublastin or a precursor of neublastin, i.e. a molecule, which in the body can easily be converted into a biologically active form of neublastin, or cells that secrete neublastin, can be additionally encapsulated, for example, in semipermeable membranes.

Cells can be grown with νίίΓΟ for use in transplantation or engraftment in the brains of mammals, including humans.

In a preferred embodiment, the gene encoding the polypeptide of the invention is transfected into a suitable cell line, for example, a line of continuously dividing rat stem cells like H1B5 and KX33b, or a line of immortalized human nerve cell precursors, and the resulting cell line is implanted into the brain of a living organism , including humans, to secrete the therapeutic polypeptide of the invention in the central nervous system, for example using expression vectors described in international patent application XV 0 98/32869.

Diagnostic and screening methods

Neublastin nucleic acid can be used to determine whether an individual is predisposed to develop a neurological disorder resulting from a defect in the neublastin gene, such as a defect in the neublastin allele, which is obtained due to genetic inheritance, due to abnormal embryonic development or as a result of acquired DNA damage. The analysis can be carried out, for example, by detecting deletions (s) or point mutations (s) within the neublastin gene or by identifying the inheritance of such a predisposition to these genetic defects based on polymorphism of the lengths of specific restriction fragments (RFLP), by detecting the presence or absence of a normal gene neublastin by hybridizing a patient's nucleic acid sample with a nucleic acid probe (s) specific for the neublastin gene, and detecting the ability of the probe to hybridize with the nucleic acid lotoy.

In particular, a neublastin nucleic acid may be used as a probe for hybridization. Such hybridization studies can be used to detect, predict, diagnose or monitor various conditions, disorders or diseases associated with abnormal levels of mRNA encoding a neublastin protein. Neublastin nucleic acid can be constructed as a marker of physiological processes dependent on the neurotrophic factor neublastin. These processes include, but are not limited to, normal physiological processes (e.g., function of neurons) and pathological processes (e.g., neurodegenerative diseases). The characterization of a particular subpopulation of patients with aberrant (i.e., elevated or deficient) levels of neublastin protein and or mRNA encoding neublastin can lead to the development of a new classification of diseases. The term aberrant levels, as used here, means an increased or decreased level relative to the level of a control sample or individual that does not have abnormalities detected by quantitative or qualitative methods.

The nucleic acids and the neublastin polypeptides of the invention can also be used for screening and identification of neublastin analogues, including small molecules of neublastin mimetics. In one contemplated embodiment, the invention provides a method for identifying a candidate candidate compound that induces a neublastin-mediated biological effect, a method comprising the steps of preparing a test cell in which expression of a detectable product is induced upon contact with neublastin, treating the selected compound for study and identification of a registered product. The expression of the registered product is an indicator of the ability of the compound selected for the study to induce a biological effect mediated by neublastin.

In addition, the nucleic acids and neublastin polypeptides of the present invention can be used in DNA chips or protein chips or in computer programs to identify related new gene sequences and the proteins encoded by them, including allelic variants and polymorphic forms that differ only in one nucleotide (SNP). Such methods are described, for example, in US patent No. 5795716, 5754524, 5733729, 5800992, 5445934, 5525464, each of which is incorporated herein by reference.

Examples

Example 1. Methods for the isolation of nucleic acids of neublastin.

Method 1. Quick screening of human embryo brain cDNA for the presence of the neublastin gene.

Fragment 290 bp long was identified in two high-performance genomic sequences (HPAI) presented in OeyVaik (Inventory Νο. AC005038 and AC005051), based on their homology with human perseffin. On the nucleic acid sequence of the 290 bp fragment two neublastin-specific primers were synthesized. The primer of the upper chain of the neublastin gene (ΝΒΝίπΙ.sense) had the sequence 5'-CST OOS CaO SST AST LLC-3 '| 8E0 GO N0: 17]. The primer of the lower chain of the neublastin gene (ΝΒΝίηΐ. Antisense) had the sequence 5'-AAO OAO ACC OST TSO TAO СО-3 '[8Er GO N0: 18]. Using these primers, 96-well PCR reactions were performed.

A 96-well main plate containing plasmid DNA of 500,000 cDNA clones was filled with approximately 5,000 clones per well. A 96-well sub-plate was used with the Ε.εοίί ΌΗ10Β glycerin culture, and it contained 50 clones per well.

Neublastin nucleic acid was identified as a result of three rounds of amplification using polymerase chain reaction (PCR) technology; amplification increased the number of copies of the nucleic acid in the sample.

Screening on the main tablet. Using the 96-well PCR screening method described above, the human embryo brain cDNA was screened on the main plate using gene-specific primers to isolate human neublastin cDNA.

From the corresponding well of the main plate, thirty nanograms (30 ng) of human embryo brain cDNA were obtained (6 ng / μl; Opdep Tesypo1od1ek) and it was introduced into the mixture with a total volume of 25 μl, which contained the following reagents: 0.2 mM of each of the above gene -specific primers (i.e. ΝΒΝίηΐ. semantic and ΝΒΝίπΙ. antisense). 1 x standard PCR buffer (Buffer V, Abuapsebe Vu1esipo1od1ek, IR), 0.2 mM dNTPs (Ategkyat-Ryattas1a), 0.1 M OC-melt (S1op1esy Laboguepek, I8A); and 0.5 units of Tad polymerase DNA (5 units / μl; Abuapseb Vuliconodonec, IR).

Thermocyclic PCR reactions were performed using the following procedure and under the following conditions. Initially, the DNA was denatured at 94 ° C for 3 min, and then 35 denaturation cycles were followed at 94 ° C for 1 min each, annealed at 55 ° C for 1 min, the first elongation at 72 ° C for 90 s; and final elongation at 72 ° C for 5 minutes The products of 96 individual PCR reactions were analyzed by gel electrophoresis using a 2% agarose gel containing ethidium bromide dye. It was found that the positive PCR product detected in the well was 102 bp in length. corresponds to a single 96 well sub-plate.

A nucleic acid fragment of 102 bp in length had the following sequence | 81T) GO Ν0: 13]:

5’-STEESSASSSSTASTEESSSSSSSEOSSSSSTESEASSESSSSSS6SSSSSSSSSSSSTS6SSAESSSTESTSSSSASSASSAS6SSTAS EAASSOOSTSTSSTT-3 '

Screening on sub-tablets. A 96-well sub-plate of the brain of the human embryo was screened by PCR amplification by placing 1 μl of glycerol culture from the corresponding well of the sub-tablet in a mixture with a total volume of 25 μl, containing: 0.2 mM of each of the two gene-specific primers ; 1 x standard PCR buffer (Buffer V, Abuapsebe Vu1esipo1od1ek, Central Committee), 0.2 mM dNTPs (Ategkyat-Ryattaaa), 0.1 M OC-melt (S1op1esy Laboguepek, I8A); and 0.5 units of Tad polymerase DNA (5 units / μl; Abuapseb Vuliconodonec, IR).

The same thermocyclic PCR conditions were used as described for screening on the main plate. 96 individual PCR reactions were analyzed on a 2% agarose gel containing ethidium bromide, and a positive well was identified from which a 102 bp PCR fragment was obtained.

Colon PCR: 1 ml of glycerol culture from the positive well of the sub-tablet was diluted 1: 100 in Luria medium (EB). 1 ml and 10 ml of the above dilution were placed in two separate tablets with agar containing Luria medium (LB), and 100 μg / ml of carbenicillin. Then, the BB plates were incubated overnight at 30 ° C. 96 colonies from these plates were collected in a new 96-well PCR plate containing 0.2 mM of each of the two gene-specific primers indicated above, 1 x standard PCR buffer (Buffer V, Ayuapsey B1o! Esbpo1od1ek, IR), 0.2 mM dNTPs (ATEGKYATRAGTAC1A), 0.1 M OC-melt (C1On! esLaLaGa! guard, I8A); and 0.5 units of DNA polymerase Tach (5 units / μl; Ayuapsey Vu! espio1od1ek, IR) in a final volume of 25 μl.

The same thermocyclic PCR conditions were used as described for screening on the main plate. 96 individual PCR reactions were analyzed on a 2% agarose gel containing ethidium bromide. Then a positive colony was identified containing a 102 bp fragment.

Sequencing of plasmid DNA obtained from this positive colony revealed 861 bp full-length cDNA. | 8EO ΙΌ N0: 8]. cDNA encoded pre-pro-neublastin | 8E0 ΙΌ N0: 9]. Automated DNA sequencing was performed using a set of terminators of the B1dEue® sequencing cycle (PE ArrNey VyukuChetk, I8A). Separation in sequencing gels was performed on ΛΕΙ Prgkt 377 (PE ArrNey Vyukuyetk, I8A).

Method 2. Cloning of human brain neublastin cDNA. An additional method for amplification of full length neublastin cDNA or a cDNA fragment can be performed using CACE (rapid amplification of cDNA ends) and neublastin-specific primers IV # p !. semantic and IV№p !. antisense, described above, in combination with primers specific for a vector or adapter, for example, using the Magafop DNA amplification kit (C1op! es baoba! oo8, u8A, Ca!. ba. K1802-1).

The ready-made complete cDNA of the human brain MagaFop (C1op! Esa baaaaaaaaaaa, IaA, Caaaaodie no. 7400-1) can be used to amplify full-length neublastin cDNA. Suitable primers for amplification include the primer of the top chain of neublastin 5'-ATOOAASTTOOASTSTOO-3 '| 8EO ΙΌ N0: 19] (No.B№x1.sense) and the primer of the lower chain of neublastin 5'-ТССАТССССССССССООС-3' | 8Е0 ΙΌ N0: 20 ] (No. В№х1.antisense) in combination with the adapter primer AP1 included in the kit with the finished Mag! Yop cDNA. An alternative primer of the upper chain 5'-СТАООАССССАТССС-3 '| 8Е0 ΙΌ N0: 28] was also used. Another alternative lower primer of the 5'-OJSCSOAOSSSTSAOOSS-3 '| 8EO ΙΌ N0: 33] can also be used. Similarly, alternative lower chain primers 8E0 ΙΌ N0: 24 and 26 can also be used.

Method 3. Cloning of human brain neublastin cDNA. Another way of cloning neublastin cDNA is to screen libraries of adult brain and human embryo using one or more of the neublast probes described herein (and as shown in the example in figure 1). These libraries include: λ§111 library of the human brain (C1op! Esy baoga! Og1ek, I8A, Ca !. No. H3002b); or λ§! 11 is the library of the human embryo’s brain (C1op! esb LabaFpek, I8A, Ca !. No. Hb3002b).

Method 4. Quick screening of mouse embryo cDNA for the neublastin gene.

The rapid screening procedure for the presence of the neublastin gene was performed as follows. A 96-well main plate containing plasmid DNA of 500,000 cDNA clones was filled with approximately 5,000 clones per well. A 96-well sub-plate was used with E.soy glycerol culture and contained 50 clones per well. Three rounds of PCR-mediated amplification were performed to identify the gene of interest (i.e., the neublastin gene).

Main plate screening: A main plate filled with purified mouse cDNA was screened by PCR in 96 wells using gene-specific primers to isolate mouse neublastin cDNA. The following two primers were synthesized:

(1) C2 neublastin primer (IV # p! Semantic): 5'-ОССАССОСССОССОАСОА-3 '| 8ЕО ΙΌ N0: 21]; and (2) C2ac primer neublastin (IV # p !. antisense): 5'-OOSOOTSSAS OOTTSTSSAO-3 '| 8EO ΙΌ N0: 22]. Using these two gene-specific primers, a positive 220 bp PCR product was identified. Nucleic acid 220 bp long had the following sequence | 8EO ΙΌ N0: 14]:

5’-SSSSSASSSSSTSSbASSASSTSATASetTTSSSSTTSTbSSASSeb

STSbTessSSSSSSASSASSASESSSSSSASSASATATSSAETSTESSAS SSTASTSSSSSSSTESSSSSTASSSTSSSSSSSSSSSTSSSSSSSSATSSASSSSSTSTSSSSSSASSSTAT6ASSSSSTST STSTATS6ASSTASASSASSASSASSASSASASSASSAS

Then, PCR reactions were performed in 96 wells as follows. Thirty nanograms of mouse embryo brain cDNA (6 ng / μl; 0dep Tes11po1od1ec) were obtained from the corresponding wells of the main plate and added to the mixture with a total volume of 25 μl, which also contained: 0.2 mM of each of the two gene-specific primers mentioned above (i.e. e. C2 primer (No. N1 ^ semantic) and C2ac neublastin primer (IV # p! antisense)), 1 x standard PCR buffer (Buffer V, Aimapsey Vyu! esypo1od1ek, IR), 0.2 mM dNTPs (ATEGKAT- Ryagtaaa), 0.1 M OC-melt (C1On! EsLaLoga! OG1EK, I8A); and 0.5 units of DNA polymerase Tach (5 units / μl; Ayuapsey Vyu! espio1od1ek, IR).

The following PCR thermal cycling conditions were used: initial denaturation at 94 ° C for 3 min, followed by 35 denaturation cycles at 94 ° C for 1 min each, annealing at 55 ° C for 1 min, elongation at 72 ° C for 90 s;

and final elongation at 72 ° C for 5 minutes 96 individual PCR reactions were analyzed on a 2% agarose gel containing ethidium bromide dye. It was found that a 220 bp positive PCR product detected in the well corresponds to a single 96-well sub-plate. Then, 96 individual PCR reactions were analyzed by electrophoresis on a 2% agarose gel containing ethidium bromide dye. The positive PCR product that was identified corresponded to a single well of a 96 well sub-plate.

Sub-plate screening: A 96-well sub-plate of the mouse embryo brain was screened by PCR-mediated amplification by placing 1 μl of glycerol culture from the corresponding wells of the sub-tablet into a mixture with a final total volume of 25 μl, which contained: 0.2 mm each of the two gene-specific primers mentioned above; 1 x standard PCR buffer (Buffer V, AITAPSEY VU1ES11po1od1e5, IR), 0.2 mM dNTPs (Ategayat-Ryattaaa), 0.1 M OSplast (С1оСес11 11ааааюпех И8А); and 0.5 units of Tad polymerase DNA (5 units / μl; Ayuapseay | O1ec11po1oDe5, IR). PCR thermal cycling was performed in accordance with the conditions described above for screening a main plate.

Then, 96 individual PCR reactions were analyzed on a 2% agarose gel containing ethidium bromide and a positive well was identified in which a 220 bp fragment was obtained.

Colon PCR: One ml of glycerol culture from the positive well of the sub-tablet was diluted 1: 100 in Luria medium (LB). Then one ml and 10 ml of the above dilution were placed in two separate LB tablets containing 100 μg / ml of carbenicillin and incubated overnight at 30 ° C. A total of 96 colonies were isolated and transferred to a 96-well PCR plate containing: 0.2 mM of each of the two gene-specific primers mentioned above, 1 x standard PCR buffer (Buffer V, Ayuapsey Vu1es11po1od1e5, IR), 0.2 mM dNTPs (Ategayat-Ryattasha), 0.1 M OS-melt (Cl1b1b2bAbO2u8I8A); and 0.5 units of Tad DNA polymerase (5 units / μl; Ayuapsei B1o1esipo1od1e8, IR) in a final volume of 25 μl.

PCR thermal cycling was performed according to the conditions described above (see screening on the main plate below). 96 individual PCR reactions were analyzed by gel electrophoresis on a 2% agarose gel containing ethidium bromide. A positive colony was identified by the presence of a 220 bp fragment. Plasmid DNA was obtained from this positive colony. The clone was sequenced using automated DNA sequencing using a set of terminators of the sequencing cycle B1dBue® (PE Arrey Vyukljetkh I8A) with DNA polymerase AtryTad. Separation in sequencing gels was carried out on AB1 Prbm 377 (PE Arréy Vüklütch I8A). In the obtained sequence of this clone, full-length cDNA from 2136 bp was detected. | 8ETS GO N0: 15]. The cDNA includes an open reading frame with the calculated amino acid sequence shown in 8EC GO N0: 16, which encodes a pre-pro-neublastin polypeptide.

Example 2. Cloning of the genomic sequence of neublastin.

As discussed above, applicants identified a 290 bp nucleic acid fragment in two IBC human clones using OepVapk elements (with inventory no. АС005038 and АС005051), which had homology regions with perseffin and with flanking persefin sequences. Applicants used 861 bp the predicted sequence described above in order to develop additional primers for the purpose of cloning a nucleic acid encoding additional neublastin nucleic acids using the Laketdepe computer program (□ CHASIA, 1ps.). Two pairs of primers were used to clone the neublastin gene using PCR reactions on genomic DNA. Two pairs of primers are shown below.

A pair of primers No. one

5'-SSA AdS SSA SST ddd tds SST STT TST SS-3 '(semantic) | 8ES GO N0: 23].

5'-CAT SAS CCA SSd dsa ddd dSS TST SAD-3 '(antisense) | 8EC GO N0: 24].

A pair of primers No. 2 5'-dadSSCA1dSSSddSSTdATSTSadSS sda ddASA-3 '(semantic) | 8EC GO N0: 25].

5'-SSSTddSTdAddSSdSTddSTADddddAS TSTdS-3 '(antisense) | 8EC GO N0: 26].

Using a pair of primers No. 1, a DNA fragment of 887 bp was amplified. from the preparation of human genomic DNA purchased in Clop (Es baotaFpec (Cat. No. 6550-1).

PCR protocol: PCR was performed using a high-precision PCR system Exrap ™ (Voyeppdet Mappepp) with buffer 1. 5% dimethyl sulfoxide (DMSO) and 17.5 pmol of each of dNTP in a total volume of 50 μl were added to the reaction mixture for PCR. Thermal cycling was carried out with a preliminary denaturation step at 94 ° C for 2 min, followed by 35 two-stage cycles at 94 ° C for 10 s and at 68 ° C for 1 min, respectively. Thermal cycling was completed by incubation at 68 ° C for 5 min. Thermal cycling was carried out in a RTS-225 DNA thermal cycler Epte Te (guy (yegtosu sel (M1 Ks5sage11. MA). PCR products were analyzed by electrophoresis in 2% agarose (EMC) and then photographed.

A fragment of 887 bp amplified on human genomic DNA using a pair of primers No. 1 was cloned into a pCID vector (1puCgodep) and used to transform competent E. colH1B1ie cells (81a1adepe). The resulting plasmid, called neublastin-2, was sequenced using a thermosequenase (Ategkyat Ryagtaa B1o! Esy). Sequencing products were analyzed by electrophoresis on an AEExrgeck automatic sequencer (Ategzyat Ryagtas1a Vu1es11).

Fragments obtained by PCR amplification of human genomic DNA using a second pair of primers (a pair of primers No. 2 above) were sequenced, and an additional region of 42 bp was revealed. at the 3'-end of the open reading frame. The full-length sequence was analyzed by comparing it with the nucleic acid sequences of other neurotrophic factors, as well as by mapping exon-intron borders using computer programs for finding genes that identify possible compounds in splicing and areas with a high probability of encoding using computer programs No. 1 Depe and Sep Magk (Vgipak e! A1., 1. Mo1. Vu1., 1991, 220, 49-65); Vogobo ^ ku e! A1., No. c1Ac1b5 Kez., 1995, 23, 3554-62). The boundaries of exons-introns were confirmed using cDNA obtained by rapid screening described above.

As shown in Fig. 7, the resulting neublastin gene has two exons separated by an intron 70 bp long. Together, the ekeons contain the predicted amino acid sequence of the full length neublastin polypeptide. The predicted cDNA | 5> EO GO ΝΟ: 3] contains an open reading frame (OPC) encoding 238 amino acid residues | 5> EO GO ΝΟ: 4]. Clone neublastin-2 contained the complete pro-neublastin coding sequence. The amino acid sequence encoded by the gene showed high homology with three proteins, perseffin, neurthurin, and SIIE.

Example 3. Expression of nucleic acids of neublastin.

The expression of neublastin RNA was detected both in nervous and non-nervous tissue in rodents and humans and at various stages of development of a young and adult organism using the methods described below.

Method for detecting neublastin RNA expression using RT-PCR

Based on the DNA sequence of neublastin designated 8EO GO ΝΟ: 1, the following primers were synthesized:

(1) neublastin primer C2 5'-SSSSACS SSSTSSSASSASAS-3 '^ ΗΕ) GO 21: 21] and (2) primer neublastin C2as 5'-SSS SSTSSASSSSTSTSTSSAS-3' [8Er GO ΝΟ: 22].

This set of primers was used for RT-PCR amplification of a DNA fragment on mRNA of a whole adult brain and human embryo. Among the DNA fragments obtained by this reaction, one was 220 bp in length. The identification of this DNA fragment with a length of 220 bp confirmed that the neublastin gene is expressed in adult and embryonic brain tissue. 220 bp DNA fragment was also amplified on genomic DNA using these primers.

Method for detecting neublastin RNA expression using Northern blot hybridization

Northern blots with polyA ⁺ RNA from adult tissues were obtained from a commercial supplier (C1op (ec11 EaLogaUpeh, u8A) and examined using a ³² P-labeled neublastin cDNA probe. Labeled neublastin cDNA was prepared according to the method described in Example 1 above .

Preparation of probes: a neublastin nucleic acid fragment (nucleotides 296-819 8EO GO ΝΟ: 8) was labeled with Kebpert II labeling kit (Integzyat; Cat. No.

KRYU633) in order to use as a probe in hybridization, as recommended by the manufacturer. Briefly, a DNA sample was diluted to a concentration of 2.5-25 ng in 45 μl of 10 mM TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Then the DNA was denatured by heating the sample to 95-100 ° C for 5 min in a boiling water bath, the sample was quickly cooled by placing it in ice for 5 min, and then it was centrifuged briefly to move the contents to the bottom of the reaction tube. The total amount of denatured DNA was added together with 5 μl of [ ³² P] dTCP Kebbie (Integrate Ryabtaa B1o! Esb L1b.) Into a reaction tube containing a buffer solution of DATP, dGTP, dTTP, an exonuclease-free Klenov enzyme and random primer in a dry, stable form. The solution was mixed by pipetting up and down 2 times, rotating the tip of the pipette in the solution, and the reaction mixture was incubated at 37 ° C for 10 min. The labeling reaction was stopped by the addition of 5 μl of 0.2 M EDTA. For use as a hybridization probe, labeled DNA was denatured to single stranded strands by heating the DNA sample to 95-100 ° C for 5 minutes, then instantly cooling the DNA sample on ice for 5 minutes. The tube was centrifuged and its contents mixed well. At the end, a single-stranded DNA probe was purified using a nucleotide removal kit (fadep).

Hybridization methods: ready-made northern blots were purchased from a commercial supplier (MiShr1e T1kkie ΝογΙΙκγπ ΒΙοΙκ, С1оп1ес11 ^ аЬο ^ аΐο ^^ еκ, И8А, Νο. According to catalog 7760-1 and 7769-1), and the hybridization was carried out in accordance with the manufacturer’s instructions, using a ³² P-labeled neublastin probe prepared as described above. For hybridization, a solution of ExrhekkNuB (SkhSeBbAa, Hakpek, I8A) was used and a concentration of the labeled probe of about 3 ng / ml was used. The solution of ExhekkNuB was heated to 68 ° C and then stirred to dissolve any precipitate. Each Northern blot membrane (10 x 10 cm) was prehybridized in at least 5 ml of ExrheccnHbn solution at 68 ° C for 30 min in a Hybalb hybridization thermostat in accordance with the manufacturer's instructions. ^{32 The} labeled neublastin probe was denatured at 95-100 ° C for 2 minutes and then rapidly cooled on ice. Fourteen microliters (14 μl) of the labeled probe were added to 5 ml of fresh Exrhecker and mixed thoroughly. The Exhecker solution used in the preliminary hybridization was replaced by uniformly distributing 5 ml of the fresh Exhecker solution containing the labeled DNA probe over the blots. Blots were incubated at 68 ° C for 1 h in an incubator for hybridization of HbA1y. After incubation, the blots were washed off and washed several times under mild conditions (2 × 88 ° C buffer containing 0.05% 8–8 at room temperature), followed by washing under severe conditions (0.1 × 88 ° C, containing 0.1% 8–8 at 50 ° C) ) [20X 88C is 0.3M No. 10 / 0.3M citrate No., pH 7.0]. Blots were exposed with a film NuregSht MR (AtegkBat RBagtas1a Β ^ ΐΐΐο....).) At -80 ° C, using reinforcing screens.

The results of experiments on Northern blot hybridization are presented in figure 1. 1A (left) and FIG. 1B (right) are Northern polyA ⁺ RNA blots that were tested with a ³² P-labeled neublastin cDNA probe, as described in Example 3. Markers are represented by nucleotides of 1.35 thousand nucleotide sizes (kb), 2.4 kb, 4.4 kb, 7.5 kb and 9.5 kb The membrane shown in FIG. 1A was obtained with mRNA extracted from various tissues of an adult. Based on the results of analysis using Northern blot hybridization, the applicants concluded that neublastin mRNA is expressed in many adult tissues. The highest level of neublastin expression was found in the heart, skeletal muscle, and pancreas. The membrane shown in FIG. 1B was obtained with RNA extracted from various regions of the adult brain. Within the adult brain, the highest level of expression is observed in the caudate nucleus and in the thalamus. A preferred form of neublastin mRNA expressed in the brain was an approximately 5 kb mRNA transcript.

A method for detecting expression of neublastin mRNA using ίπ kin hybridization in tissues

The following methods were used to measure the expression of neublastin RNA in animal tissues, such as rodent tissues, using an antisense neublastin probe.

Expression in Mice

Preparation of tissue samples: mice (B & K Ishuegka1, ZkskMt, 8 \\ 'nyep) were killed during pregnancy by displacing the cervical vertebrae by 13.5 or 18.5 days of pregnancy. When opening under sterile conditions, embryos were removed and immediately immersed in a solution of 0.1 M phosphate buffer (PF) containing 4% paraformaldehyde (PFA) for 24-30 hours and then removed from PFA and stored in PBS (phosphate-buffered saline) . The tissue was prepared to obtain sections by immersing the tissue in a solution of 30% sucrose and then enclosing it in T1kkie TesP blocks (O.S.T. Sotroty, 8akiga Eteke I8A, SA). Six series of coronary or sagittal sections (12 μm each) were made in a cryostat and thawed by placing on positively charged glass slides. The heads / brains of newborns (P1, P7) were fixed, following the same protocol as for the embryonic stages, and adult brain tissue was dissected, immediately frozen on dry ice and sections were made in a cryostat without any preliminary blocking.

Preparation of neublastin ribosondes: an antisense neublastin RNA probe (below the neublastin ribosonde) was prepared as follows. The nucleotides 1109-1863 of the mouse neublastin cDNA sequence [8EC ΙΌ ΝΟ: 15] were subcloned into the Β1е8с ^^ рΐ vector (§1ga (adeche). The resulting plasmid was cut to obtain linear DNA using the restriction endonuclease ΕсоК1. Etok! DNA fragment was transcribed ί νίίτο using T3 RNA polymerase and a kit for RNA labeling with digoxygenin (DIG) in accordance with the manufacturer’s instructions (Б Б Б г д д М ап)))).

Hybridization: cryostatic sections were fixed for 10 min in 4% PFA, treated for 5 min with 10 mg / ml proteinase K, dehydrated sequentially in 70% and 95% ethanol for 5 and 2 min, respectively, and then allowed to air dry . Hybridization buffer (50% deionized formamide, 10% dextran sulfate solution, 50% concentration, 1% Denhardt solution, 250 μg / ml yeast tRNA, 0.3 M No. C1, 20 mM Tris-HC1 (pH 8), 5 mM EDTA, 10 mM NaPΟ ₄ , 1% sarcosyl) containing 1 μg / ml DIG-labeled probe was heated at 80 ° C for 2 min and applied to sections. Then, the sections were coated with parafilm and incubated at 55 ° С for 16–18 h.

The next day, the sections were washed under high stringency conditions (2 x BBS containing 50% formamide) at 55 ° C for 30 min and then washed in RNase buffer and incubated with 20 μg / ml RNase A for 30 min at 37 ° C . To detect a DIG-labeled probe, sections were preincubated in blocking solution (PBS containing 0.1% Tween-20 and 10% heat-inactivated goat serum) for 1 h and then incubated overnight at 4 ° C with anti-DIG antibodies, combined with alkaline phosphatase, at a dilution of 1: 5000 (Woeygtdeg Mappet). The next day, for each slice, four two-hour rinses were performed in an FSB containing 0.1% tween-20, and then two ten-minute rinses were performed in buffer TMT (100 mM No. C1, 100 mM Tris-HC1 (pH 9.5), 50 mM MDS1 ₂ , 0.1% tween-20). Sections were then incubated in a VM-violet substrate containing 0.5 mg / ml levamisole for 48 hours. The color reaction was stopped by washing in PBS. The sections were dried in air and covered with a cover glass with S (KEB0-1ab, B \\ e6ep).

The results of the reactions of w κίΐιι hybridizations are presented in table. one.

Table 1. The expression of neublastin in mice

Structure E13.5 E18.5 P1 P7 Adult body Forebrain ++ Ventral part of the midbrain - Dorsal Root Ganglia ++ Spinal cord + Retina +++ +++ + Olfactory share ++ ++ ++ Tooth pulp ++ ++ + Trigeminal ganglia ++ ++ ++ Striped body + + ++ Cortex ++ ++ ++ + Jagged gyrus ++ +

As shown in the table. 1, on 13.5 fetal days (E13.5), neublastin was expressed in the spinal cord and in the hindbrain and weakly in the forebrain. The expression of neublastin was also detected in the developing retina and in the sensitive ganglia (dorsal root ganglia and trigeminal ganglion (V)). Outside the nervous system, a weak signal was detected in the kidney, lung, and intestines, suggesting that neublastin is also expressed in these tissues.

On 18.5 embryonic day (E18.5), neublastin was more pronounced in the ganglion of the trigeminal nerve (V). The expression of neublastin was also detected in the retina, striatum and cerebral cortex. In addition, expression was observed in the tooth germ.

Referring again to the table. 1, increased expression of neublastin from the time point corresponding to E18.5 to postnatal days 1 and 7 was observed in the cerebral cortex, striatum and trigeminal ganglion (V). The expression of neublastin was more noticeable in the outer layers of the cerebral cortex than in the inner layers of the cerebral cortex. On P7, expression was found in the same structures as on day 1, and additional expression of neublastin was found in the hippocampus, especially in the dentate gyrus and cerebellum. In the brain of adult mice, neublastin was strictly expressed in the dentate gyrus at very low or unregistered levels of neublastin expression in other test tissues.

Rat Expression

The following experiment describes the hybridization of rat tissue with an oligodeoxynucleotide neublastin antisense probe labeled with alkaline phosphatase.

Preparation of tissue samples: rat embryos (E14) were obtained from pregnant rats M1k1ag (Mo11eda6 Vgeebshd, Eptagk) after anesthesia with pentobarbital. Rats in the postnatal period (P0, P7, adults) were killed by decapitation. An open brain and whole heads were immediately immersed in cold 0.9% No. C1, freshly frozen and 20 μm sections were prepared in a cryostat (coronary and sagittal sections, 10 series).

1p 811 and hybridization: Two series of sections were hybridized using an antisense oligodeoxynucleotide probe conjugated to alkaline phosphatase (alkaline phosphatase) (5'№А СОТ СОТ ССС ТСС ССС ССС ССС АСС АСС С-3 '[NEC ΙΌ N0: 27], 011§ No. 164675, ^ NA Tesyio1odu, Optagk). This probe is complementary to bases 1140 to 1169 mouse neublastin cDNA BEC ΙΌ N0: 15.

Before hybridization, the sections were dried in air at room temperature, heated to 55 ° C for 10 min, and then treated with 96% ethanol at 4 ° C overnight. Then, the sections were dried in air and incubated in a hybridization medium (5.0 pmol of probe / ml) overnight at 39 ° C (Rshkep e1 a1., No. Igoksk, 1992, 47, 105-113; \\ ek! E! A1., 1. Sotr.№igo1., 1996, 370, 11-22).

The post-hybridization treatment consisted of four thirty-minute washes in 1 x BBS (0.15M No. C1, 0.015M citrate No.) at 55 ° C followed by three ten-minute washes in Tris-HC1, pH 9.5, at room temperature before application of the developer Alkaline phosphatase. The alkaline phosphatase developer was prepared immediately before use, and it contained nitros blue tetrazolium CBT, Bshta), 5-bromo, 4-chloro, 3indolylphosphate (BC1P, B1dta) and Tris-HC1-MDCl ₂ buffer, pH 9.5 (Rschep e1 a1. No. Igoksk, 1992, 47, 105-113). The manifestation of alkaline phosphatase was carried out in the dark at room temperature for 48 hours. The color reaction was stopped by washing sections in distilled water. Sections were dehydrated with gradient washings with acetone, softened in xylene phenol creosote (A11c11et, IR), purified in xylene and covered with coverslips using Epix (B1e & Vetren, Eptagk).

Control reactions consisted of (1) pretreatment of RNase-A sections (50 μg / ml, Rkagtaaa, 8 ^ uep) before hybridization; (2) hybridization of sections with a hundredfold excess of unlabeled probe; and (3) hybridization of the slices only in the presence of one hybridization buffer. The results of hybridization reactions are presented in table. 2.

Table 2. The expression of neublastin in rats

Structure E14 P0 / P1 P7 Adult body Forebrain ++ Ventral part of the midbrain - Dorsal Root Ganglia ++ Spinal cord + Retina + Olfactory share (+) ++ ++ Cerebellum + ++ + Trigeminal ganglia ++ ++ Striped body + + (+) Cortex (+) ++ ++ + Hippocampus (+) ++ ++

In rat embryos on day 14 of embryonic day (E14), neublastin was weakly expressed in the forebrain, in the hindbrain and in the spinal cord. Neublastin mRNA was also detected in the eye (retina), dorsal root ganglia, trigeminal ganglia (V) and in the kidneys, lungs, heart, liver and intestines. In newborn rats (PO), noticeable expression of neublastin was in the cerebral cortex and striatum. Neublastin expression was also detected in the olfactory lobe and in the hippocampus. In 7-day-old rats (P7), neublastin was expressed in the cerebral cortex, striatum, olfactory lobe and cerebellum. A noticeable signal was visible in the hippocampus. In adult rats, very low or unreported levels of neublastin expression were detected in most brain regions. Weak signals were detected in the thalamic nucleus, and marked expression of neublastin was detected in the hippocampus.

Example 4. Polypeptides of neublastin.

An open reading frame or coding region (CDF), designated 8ETC ΙΌ N0: 8, encodes a pre-pro-polypeptide (called pre-pro-neublastin). The amino acid sequence predicted based on the open reading frame is shown in 8EC ΙΌ N0: 9. Three variants of neublastin polypeptides were identified based on 8EC ΙΌ N0: 9. These options include:

(ί) a polypeptide, referred to herein as ^ No. N140, which has the amino acid sequence indicated in 8EC ΙΌ N0: 10;

(ίί) a polypeptide, referred to herein as ^ No. N116, which has the amino acid sequence indicated in 8EC ΙΌ N0: 11; and (ίίί) a polypeptide, referred to herein as ^ No. N113, which has the amino acid sequence indicated in 8EC ΙΌ N0: 12;

Similarly, based on the coding region (CDD) indicated in 8EC Е N0: 3, which encodes a pre-pro polypeptide having the amino acid sequence (indicated in 8EC ΙΌ N0: 4), three variants of neublastin were identified. These options include:

(ί) a polypeptide that has the amino acid sequence indicated in 8EC ΙΌ N0: 5;

(ίί) a polypeptide that has the amino acid sequence indicated in 8EC ΙΌ N0: 6; and (ίίί) a polypeptide that has the amino acid sequence indicated in 8EC ΙΌ N0: 7;

Based on the multiple sequence alignment, which is based on C1 and 8 (a1 V (1.75), 8EC ΙΌ N0: 9 was combined with the amino acid sequences of SOXE, perseffin, and neurturin. This combination is shown in Table 3.

Table 3. Comparison of the amino acid sequence of neublastin with persefin, neurturin and SECE

Full-sized neyoturin -------------------- M0VIKAAAEA5US35U51IMSK £ СЬЬ £ НВСРА Neublastin MESSEZSNZRIRP0RAJIRTIAJAE53UAEAZ55ARK5PAR £ <ESRRP Full Size Perseffin Full size 3ΡΝΓ person ----- MKENRUUAUUSEEUENTAZAGRAESKARRARARARARUZERAARGAEZZRZ Full-Size Neurthurin LITTLE JRK.TOAK1AVABAOUYAJOSAROAMEH & EETRNASYARRSRAVNAS ₽ayk Neublastin UIA8RASNR2SKTAKNSSZSRAKKRRR <2R5KRARRRRARRZA1.RR.SSB1AAEASSRSS Full Size Peosefin -MASKUESESEEE5Y2ES-0SNSRRAYASURUARESEGZZE <2 Full size 3ΡΝΓ person ΝΜΡΕΡΥΡΡΟΓΡΡνΜΡΕΙΩΑΤΙΚΒΕΚΕΒΡΡΚαΜΑνΕΡΡΚΕΡΝΡΟΑΑΑΑΝΡΞΝΕΚεκε Full-sized Neurthurin KARAKZAKRZREYEUUZEZUAZOETUGYAUSAOASEAAAAEUUOSKR Neublastin ZVAVAAZAASVVSEZEURUAAESESNVRESREUVGKGSZESZYAK-AVZRNREZEAZ Full Size Perseffin ARRNAEZSSROSNZZUAEZAUAZEEEKUTTUSASASZSRR.OAKTONSIA'AR. Full size 3ΡΝΓ person VVZVSKMKSUETA1NON-SUSTERNESSE1a ¥ SZSZSRA-AETTURK1EKI * *; * * “* ::. *:“ ·; *: *: * ’* Full-Size Neurthurin ЕЯ0ВВРЕРВЕР-ЯУЯАйРССМ> TAUEREUZGERAN8VUNTUNE5AVESASU- Neublastin LYZAZAEVRRRSZVRUZORSSKRTVUE-AUZEMRUMZTIVTURVEZATASSESS Full Size Perseffin E <2 <ZOSEANZS -------- RCCNRTVUT-RUAERRVNKMOKEROEAAASOSBS Full-size SEM · ^{- of the} person EZYAYAUZR ---- KUS0ASSKR1AGRRRE8HERRYUUUN1EVKNZAKVSSS1- *. *. *

* shows provisions that have a separate completely conservative residue;

: shows that one of the following strict groups is completely conservative: - 8TA, \ ECL NН ^ К, N ^ Е ^, RNCC, ΜΙβν, МШЕ, НУ, ЕУ ^;

• shows that one of the following less stringent groups is completely conservative: - C8A, ATO, 8AC, 8TNK, 8TPA, 8CN, 8N ^ E ^ K, \ D) E (,) NK_NrNRK, νΠΜ, ΗΕΥ.

Based on the combination of amino acid sequences shown in table. 3, it can be seen that neublastin has seven conserved residues at positions that are conserved within the сем-β superfamily. In one embodiment, the preferred neublastin polypeptide contains (seven) cysteines that are conserved at 8EO ΙΌ N0: 2 at positions 8, 35, 39, 72, 73, 101 and 103, or at 8E0 ΙΌ N0: 4 and 9 at positions 43, 70, 74, 107, 108, 136 and 138. It is known that these seven conserved cysteine residues in the над-β superfamily form intramonomer disulfide bonds (assumed, for example, in 8Е0 ΙΌ N0: 2 between cysteine residues 8-73, 35 -101 and 39-103, and, for example, in 8Е0 ΙΌ N0: 4 and 9 between cysteine residues 43-108, 70-136 and 74-138) and one disulfide bond between monomers (assumed, for example, in 8Е0 ΙΌ N0: 2 m between cysteine residues 72-72, and, for example, in 8Е0 ΙΌ N0: 4 and 9 between cysteine residues 107-107), which together with the extended beta region of the filament forms a conservative structural motif of the ства-β superfamily. See, for example, Jaort et al., Rgsjetk, 1993, 17, 176192.

Based on this sequence alignment, it was shown that neublastin is a member of the OI number of the subfamily of neurotrophic factors (E6L6-EK (¥ / Е) С8б8сРхССВР-8АххСОС, the fingerprint of the subfamily is emphasized in Table 3).

The homology of neublastin and other representatives of the OI№ family was calculated and the results are presented below in table. 4.

Table 4. Homology of neublastin polypeptides with other members of the family

Mature protein HBN140 Mature protein HBN113 Homology Homology of full-size peptides Homology Homology of full-size peptides Neurotrophic factor Identity Overlapping (ak) Strict homology Identity Identity Overlapping (ak) Strict homology Identity (And) ^ ' 34% (47/137) 137 48% (67/137) 31.9% 36% (41/111) 111 52% (59/111) 29.5% NΤN 48% (61/127) 127 56% (72/127) 36.9% 49% (56/114) 114 57% (66/114) 44.7% P8P 44% (55/125) 125 56% (71/125) 36.9% 45% (51/111) 111 57% (65/111) 44.3% SHA 31% (25/81) 81 - 25.2% 31% (25/81) 81 - 22.5% ΤΟΕ-β2 23% (17/73) 73 - 18.5% 23% (17/73) 73 - 20.2%

= neurotrophic factor derived from the glial cell line. NΒN = Neurturin

P8P = Persefin

ΙΗΑ = inhibin-α

ΤΟΕ-β2 = transforming growth factor - β2.

Strict homology indicates that the following strict groups are conservative: 8ΤΑ, MPA MJAA MAO, ΟΗΠΕ, ΜΙΕν, ΜΙΕΕ, ΗΥ, ΕΥΑ.

Example 5. Obtaining neublastin.

The authors obtained neublastin in both types of eukaryotic and prokaryotic cells, as described below.

Expression vectors

The full length cDNA encoding neublastin was inserted into the eukaryotic expression vector ρΌΝΙΖ. This vector was created by cloning the human ILI promoter into a modified version of ρc ^ NΑ3.1 / Ζеο. Unmodified ρс ^ NΑ3.1 / доступенеο is available for commercial purchase (ΙιινίίΓοdep). The modified ρс ^ NΑ3.1 / Ζеο is smaller than the original vector, since the ampicillin gene (positions 3933 to 5015) and the sequence in positions from 2838 to 3134 were removed from it. In this modified version, ρс ^ NΑ3.1 / Ζеο the CMV promoter was replaced by the promoter of ILS from ρΤΤΙ-8 (1st appendix eί a1., EEB8 BeI., 1990, 267, 289-294), resulting in ρυόίΐζ.

Mammalian cell expression

Then, the ρυόίΙΖ vector containing the coding sequences of neublastin was transfected into the mammalian cell line H1B5, which is an immortalized rat nerve cell line (WepGgaph e A1, Ce11, 1991, 66, 713-729). Several H1B5 cell lines were created that stably express neublastin (as determined using RT-PCR). In one of these stable cell lines, Η ^ B5ρυЬ ^ 1ζNВN22, expression was confirmed by hybridization of total RNA on a Northern blot with a neublastin probe labeled ³² P. The results of these studies are shown in FIG. 2. Then

Н1В5риЫк№№2 was used as a source of neublastin to study the neurotrophic activity of neublastin.

In FIG. Figure 2 shows the expression of neublastin cDNA in the H1B5riK2 # 2 clone (i.e., Northern blot analysis using ³² P-labeled neublastin cDNA of the present invention, as described below). The blot was prepared using total RNA extracted from untransfected H1B5 cells, H1B5pri1x cells

HBN22 and H | B5pb | 1xCEXP 14, respectively, as shown. On the blot, the positions of 288 and 188 rRNA bands corresponding to 4.1 kb are indicated. and 1.9 bp, respectively.

As shown in FIG. 3, antibodies induced against polypeptides of neublastin derivatives also recognized a protein of approximately 13 kilodaltons (KD) in the conditioned medium of clone H | B5riL1xHB # 2. but not in non-transfected H1B5 cells (compare Example 6).

The predicted molecular weights of unmodified (that is, in the absence of post-translational modifications) neublastin polypeptides НВN140 | 8ЕО ГО ΝΟ: 10], ΝΗΝ116 | 8ES) GO ΝΟ: 11] and IWM 13 | 8ES) GO ΝΟ: 12] were determined 14.7 kilodaltons (kD), 12.4 kD and 12.1 kD, respectively.

Ways

Northern blot with total RNA (10 μg) from non-transfected H1B5 cells and clone H | B5riY1xHB # 2 was prepared by electrophoresis in 0.8% formaldehyde-agarose gel and blotting on a nylon membrane (Eigavpe, 81 gaadepe). The blot was hybridized and washed as described in Example 3 using a ³² P-labeled probe 1.3. N. obtained by random labeling, covering 8EO GO ΝΟ: 8 and additional nucleotides from 5'NTR and 3'NTR cDNA of neublastin. The blot was exposed with Nuregst MR (Ategkyat) at -80 ° C using amplifying screens.

The conditioned medium from H | B5pri1xHB # 2 cells or non-transfected H1B5 cells incubated overnight in serum-free medium supplemented with Ν2 supplementation (Bbbcr; cheek; Ca!. 17502-048) was concentrated and separated on 15% polyacrylamide gels (Ategkat Ryagtaaa Vu! Yes; Sa !. Νο. 80-1262-01). Proteins were transferred to RUER membranes (ATEGKAT Ryagta Da Vu! Es; Ca !. Νο. ΚΡΝ-303Ρ) and non-specific protein binding sites were blocked with 5% skimmed milk powder in FSB with 0.1% tween-20. The membranes were incubated overnight with polyclonal neublastin antibodies (1: 1000), after which they were incubated with the second antibodies against rabbit 1dC (Atebbat Papillon Va! B; Ca !. No. 934) conjugated to horseradish peroxidase (1: 2000). Immunological staining was visualized using enhanced chemiluminescence (UHL) (ATEGKAT PAGTAA VU! Es; Ca !. Νο. ΚΡΝ2109) or UHL ⁴ (ATEGKAT PAGTAAA VU!;; Ca !. .ο. ΚΡΝ2132) according to the manufacturer’s instructions (according to the manufacturer’s instructions). .

The results of these experiments are shown in FIG. 3. FIG. 3A and 3B illustrate the expression of neublastin protein in transfected H1B5 cells. Medium from overnight non-transfected H1B5 cells [lane 1] or from H1B5 clone stably transfected with neublastin cDNA [lane 2] was concentrated as described below. Then the medium was analyzed using Western blotting using two different types of polyclonal antibodies At-1 and At-2, described in example 10, specific for neublastin. It was found that in the medium obtained from transfected cells, both types of antibodies recognize a protein with a molecular weight of approximately 15 kD. This protein is not detected in untransfected H1B5 cells.

The cloned cDNA encoding neublastin can also be inserted into another eukaryotic expression vector, for example, into the eukaryotic expression vector TE1-8 (^ ο а η ке ке η е e! A1., REV8 е e !!., 1990, 267, 289-294) or pcEXA- 3 (1n1! Yeardep), and the resulting expression plasmid can be transfected into an alternative mammalian cell line, for example, Chinese hamster ovary (CHO) cells, HEK293, CO8, PC12 or KH33b cell lines or human stem nerve cells. Stable cell lines expressing neublastin are used, for example, to produce neublastin protein.

Expression in CHO cells

Construction of the plasmid p1C070.14. To express neublastin cDNA in Chinese hamster ovary cells, a cDNA fragment encoding the pre-pro form of human neublastin was inserted into the mammalian expression vector pEAC347 to create plasmid p1C070.14. pEAC347 contains the tandem of the early promoter 8U40 and the main late promoter of the adenovirus (obtained from the plasmid pAE2beta; ΝοΠοη apb ΓοΓΓίη, Мo1.Се11. В ^ ο1., 1985, 5, 281), a unique cloning site Νο! 1, followed by a late term signal 8U40 transcription and polyA signal (obtained from plasmid pCMU beta; MacSte ^ apb Sakkeu, Schc! Aslk Kek., 1989, 17, 2365). In addition, pEAC347 contains the backbone obtained from the plasmid pIS19 and the 6Si gene obtained from p8U26Yg for selection in the presence of methotrexate (MTX) and amplification in transfected CHO cells.

Plasmid p1C070.14 was created in two stages. First, the fragment encoding the pre-pro forma of human neublastin was isolated from the plasmid pU-XBX using the polymerase chain reaction with ΚΌ2- oligonucleotides

824 5'AASSAAAAAAASSSSSSSSSSSA TSSAA STTSS ASTTSSASS3 '| 8ES) GO ΝΟ: 31], KGO2-

825 5'TTTTTTSSTT 66C66CC6ST SASSSSSSSSSSSSSSSSASS3 '| 8ES) GO ΝΟ: 32] and PPi polymerase. The fragment was cloned into the 8gG1 site of the rRSC-8spr! Atp 8K (+) to create plasmid p1C069. At the second stage, partial digestion of !ο! -1 of plasmid p1C069 was carried out to form a fragment of length 685

bp (containing the neublastin gene) that was cloned into the Νοΐ-1 site of plasmid pEAC347 to create plasmid p1C070.14. Transcription of the neublastin gene in plasmid p1C070.14 is controlled by the main late promoter of the adenovirus.

Creation of CHO cell lines expressing neublastin. 200 μg p1C070.14 was linearized by digestion with restriction endonuclease M1i-1. DNA was extracted with phenol: chloroform: isoamyl alcohol (25: 24: 1) and precipitated with ethanol. The linearized DNA was resuspended in 20 mM Nerek pH 7.05, 137 mM NaCl, 5 mM KCl, 0.7 mM Ν ^ ΡΟ ^ 6 mM dextrose (HEB8) and introduced into ~ 4E7 CHO bikh B1 (bGrg) cells (p23) by electroporation (280 V and 960 μF). After electroporation, the cells were returned to the culture in a + Eagle’s modified medium (ISI) supplemented with 10% fetal bovine serum (FBS) for two days. The cells were then trypsinized and re-plated in 100 mm plates (100,000 cells / plate) in α-MSI (in the absence of ribo- and deoxyribonucleosides) with the addition of 10% dialyzed PBS for five days. Then the cells were divided to a density of 100,000 cells / 100 mm dish and selection was performed in 200 nM methotrexate. Resistant colonies were selected and expanded in 6 well plates; the conditioned medium from each clone was screened using the specific neublastin assay described below. Twelve clones expressing the highest level of neublastin were expanded in T162 vials and then re-examined. As shown in FIG. 10, CHO cell lines produced neublastin ranging from 25 to 50 ng / ml.

The study of neublastin by analysis of the triple complex. The investigators examined the presence of neublastin in the medium from supernatants of the CHO cell line using a modified analysis of the triple complex described in Sachco1a! a1. (Proc. No.! 1.Asab.8cEi8A, 1997, 94, 6238).

In this study, the capabilities of ΟΌΝΕ-like molecules can be assessed by their ability to mediate binding between the extracellular domain of BET and various co-receptors, ΟΕBα1, ΟΕBα2 and ΟΕBα3. Soluble forms of BET and co-receptors were obtained as fusion proteins. A hybrid protein formed by the extracellular domain of rat BET and placental alkaline phosphatase (BET-ALP) and a hybrid protein between the extracellular domain of ΟΕΡα.1 rats (claimed in published application νΟ 9744356; November 27, 1997, incorporated herein by reference) and Εc domain 1dC1 human (ΎΟΕΡαΓΙβ) are described (8apaso1a e! a1., Proc. No. 11. Asab.8sSh8A, 1997, 94, 6238).

To create a fusion protein between the extracellular domain of βBα3 of the mouse and the β1 domain of human 1dC1 (tBB3-Id), a DNA fragment encoding amino acids 1-359 of BETB3 mice was ligated with a fragment containing the βc domain of 1dC1 of a person and cloned in a pECC347 expression vector to create plasmids RSL44. Plasmid pCL44 transfected Chinese hamster ovary (CHO) cells to obtain a stable cell line producing a hybrid protein that was purified on an immunoaffinity column containing L-Sepharose protein using standard methods. In general, if in this study a ΟΌΝΕ-like molecule can mediate the binding of the co-receptor to BET, then the BET-ALP fusion protein will be retained on the plates, and the amount that will be retained can be measured using a chemiluminescent alkaline phosphatase substrate.

The Eupech M1sgo1ye-1 EB18A tablets (Eupech Tes11po1od1k) were coated with goat antibodies specific for human Ε s at a concentration of 1 μg / ml in 50 mM bicarbonate / carbonate, pH 9.6, for 16 hours. The contents of the tablets were drained and filled with 300 μl 1% Ι-blocking agent (Thor1x) in TBS / 0.5% tween-20 (TBST) for 1 h. After washing the TBST three times, the wells were filled with 100 μl 1 μg / ml (CBA ^ d or tΟΕBα3-Id diluted in conditioned medium of 293EΒNA cells expressing the BET-ALF fusion gene 100 μl of conditioned medium from neublastin CHO clones was added to the upper cells well th column and two-fold serial dilutions made in each bottom row of wells and incubated for 1.5 hours at room temperature. Plates were then washed three times with TBST, and twice with 200 mM Tris, pH 9.8, 10 mM MdS1 ₂ (Buffer C8PO) Then, the washing solution was replaced with 425 μM C8PO (Tgor1x) in C8PO buffer containing 1 mg / ml dark blue chemiluminescence enhancer (Tgor1x) and incubated for 30 'at room temperature. The yield of chemiluminescence was measured using an Oup! Ls luminometer.

In initial experiments, the authors examined whether neublastin-producing CHO cell lines could mediate the binding of ΟΕBα1 or ΟΕBα3 to the extracellular domain of BET. As shown in FIG. 11, the conditioned environment of the SNO cell clone No. 53 gave a strong signal in the study of the triple complex when the tΟΕBα3-Id fusion protein was turned on, but did not give a signal when the ιΌΕΡα1χ fusion protein was turned on, which indicates that neublastin binds to ΟΕBα3, but not with ΟΕBα1. This behavior clearly distinguishes neublastin from ΟΌΝΕ; as shown in FIG. 11, ΟΌΝΕ binds to CEK-1, but does not bind to CEK-3. No signal was observed with any hybrid co-receptor proteins when examining the conditioned medium of the initial CHO cell line or pure medium.

In order to quantify the levels of neublastin expression in CHO cell lines, a standard curve was obtained using gCEKaYd and SENA starting at a concentration of 1 ng / ml. Then, using a standard curve, neublastin concentrations were calculated for various CHO cell lines; the levels produced by the five CHO cell lines are shown in FIG. 10. Since this definition depends on an unverified assumption that the binding affinity between COOT ¹ and CEK- ¹ is similar to the binding affinity between neublastin and CEK-3, these levels are only approximate.

Analysis of neublastin from supernatants of CHO cell lines

In order to further analyze the neublastin produced by CHO cell lines, a protein was extracted from the medium using CEA-3-1d fusion protein and analyzed using Western blots using polyclonal antibodies obtained against neublastin peptides.

In the first experiment, neublastin was extracted with tCEAA3-1d attached to sepharose beads. tCEYAa3-1d was attached to Sepharose beads under the conditions recommended by the manufacturer, Rbagtas1a 1ps. 100 μl of tCEAA3-1d-Sepharose was added to 1.0 ml samples of conditioned medium from the CHO cell line, which is a negative control, or from the CHO cell line No. 16 producing neublastin. Suspensions were incubated for two hours on a rocking chair. Each suspension was centrifuged and the supernatant was removed after three washes of 1.0 ml of 10 mM HEPE8, 100 mM No. C1, pH 7.5. In each case, the resin was resuspended in 100 μl of 2X sample recovery buffer and heated to 100 ° C for 5 min. 20 μl of sample buffer supernatant and 10 μl of molecular weight standard (EMC) were applied to each well of 10–20% of the prepared 8E8-PLAG gel (Ον1 8c1epOPs). Gel electrophoresis was carried out at a constant current of 40 mA for 72 minutes.

For Western blot analysis, the proteins were transferred by electro-blotting to nitrocellulose (8cBe1b run apb 8bbb11) in a HoGeg 8cbbbb instrument in a buffer system containing 10 mM CAP8, 10% methanol, 0.05% 8Ό8, pH 11.2 (45 min at constant current) at 400 mA). After the transfer, the nitrocellulose filter was removed from the cartridge and the molecular weight markers were visualized by staining with a solution of 0.1% red 8 in 1% acetic acid for 60 s. The membrane was cut into two parts and excess dye was removed by gentle swaying in distilled water. Membranes were blocked in a 2% solution of skimmed milk powder in TBS overnight at 4 ° C. Membranes were incubated separately with two affinity-purified antibodies against the neublastin peptide (K.30 and K.31) at a concentration of 1.0 μg / ml in a 2% solution of skimmed milk powder in TBS. Membranes were washed three times for ten minutes in TBS-tween and incubated with a goat anti-1dC conjugate of rabbit-NKR (Vyugab) conjugate at a dilution of 1: 5000 for 30 minutes. Membranes were washed three times for 10 min with TBS-tween and developed using a UHL substrate (Ategkbat). As shown in FIG. 12, specific bands were detected in the case of proteins extracted from CHO cell line producing neublastin using both antibodies (lanes 2 and 4) when compared with the bands observed in proteins extracted from the cell line serving as a negative control (lanes 1 and 3).

The type of protein below has a molecular weight of approximately 13 kD and is likely a mature neublastin domain that has arisen after cleavage of the prodomain. Such a cleavage could occur after any of the three residues of Agpre-protein of neublastin (for example, -KHKHK1-), giving either 140AK, 116AK, or 113AK forms specified in 8EC ΙΌ ΝΟ: 10, 11 or 12, respectively. The predicted molecular weights of unmodified (ie, without post-translational modifications) of the neublastin polypeptides ΝΒΝ140 | 8ЕО ΙΌ ΝΟ: 10], ΝΒΝ116 | 8ЕО ΙΌ ΝΟ: 11] and ΝΒΝ113 | 8ЕО ΙΌ ΝΟ: 12] were determined to be 14.7 kD, 12.4 kD and 12.1 kD, respectively. Additional analysis is needed to confirm the structure of these species, as well as other neublastin-specific bands.

In the second experiment, neublastin was extracted using NSEKaL-U fixed on E8A plates. In order to obtain a fusion protein between the extracellular domain of human CEK-3 (claimed in published application ^ Ο97 / 44356, November 27, 1997, incorporated herein by reference) and the Ec domain of human! DC1 (bCEK-3-! D), a DNA fragment encoding amino acids 1-364 , Human CEK-3 was ligated with a fragment containing the Ec domain of human! DC 1 and cloned into the expression vector CH269 described by 8atco1a! a1. (Proc. No.! 1.Asa6.8sEi8A, 1997, 94, 6238). The fusion protein encoded by this plasmid was temporarily expressed in cells with a nuclear antigen encoded by the Epstein Barr 293 virus (ЕΒNА), and it was purified on an immunoaffinity column with proteinA-sepharose by standard methods.

Six wells of a 96-well plate were coated overnight at 4 ° C with goat anti-human 1dC antibodies (specific for the Esu fragment; 1askzoi 1thio1od1cc) at a concentration of 25 mg / ml in PBS (300 μl / well). Wells were blocked for 1 h at room temperature with 400 μl of 1% BSA in PBS. After three washes with FSBT (FSB + 0.05% Tween20), 300 ml of ISEC-31d (10 mg / ml in FSB containing 0.1% BSA) was added to each well. The plate was incubated for 1 h at room temperature and gently shaking (200 beats / min) to ensure maximum binding. Then the wells were empty and again washed 3 times with FSBT. 250 μl of conditioned medium from the CHO cell line serving as a negative control or from the CHO cell line No. 25 producing neublastin was added to each of 3 wells. The plate was incubated for 3 hours at room temperature and gently shaking (300 beats / min). Then the wells were washed twice with PBS. 25 μl of non-reducing Laemmli application buffer was added to the first well and the plate was shaken rapidly for 5 min to elute the bound proteins (1300 bpm). The contents were transferred to the next well and the procedure was repeated to elute the proteins bound in the second and third wells. After addition of β-mercaptoethanol (5% final concentration), the samples were boiled for 5 min and analyzed using 8O8-PAG in a 10–20% polyacrylamide gel.

For Western blot analysis, proteins were transferred to nitrocellulose. The membrane was blocked and analyzed in a 5% skimmed milk powder solution, FSBT and washed in FSBT. Neublastin was detected by electrochemiluminescence after reaction with polyclonal antibodies (K30 and K31) directed against two neublastin peptides (at a concentration of 1 μg / ml), followed by reaction with goat antibodies against 1d rabbit conjugated with NKR (Vyukab). As shown in FIG. 13, five neublastin-specific bands were detected in proteins extracted from the CHO cell line producing neublastin (lane 2). The two lower bands are very similar to those seen in FIG. 12; and again, the lower band appears to be a mature neublastin domain that arose after cleavage of the pro-domain.

Subsequent analysis (data not shown) of the bands in FIG. 13 showed that deglycosylation of the PCNase E protein in a band corresponding to about 18 kDa restores the protein in that band to a size equivalent to the lowest band in the gel in FIG. 13. This confirms that neublastin can be produced in mammalian cells as a glycosylated protein.

The expression of neublastin in E. soi

To express the neublastin gene in E. soi, syngens with a lower SS content and preferred E. soi codons were constructed. Syngen is cloned in two vectors, pET19b and pM1B164, a derivative of pET19b. The construction with pET19 is shown in FIG. 14. In this construct, the sequence encoding the mature domain of neublastin (No. N113) is directly fused to the initiating codon of methionine. The design with pM1B164 is shown in FIG. 15. In this construct, the mature domain of neublastin is fused to a histidine tag (ie, 10 histidines) separated by an enterokinase cleavage site. The initiating methionine precedes the histidine tag.

The nucleotide sequence encoding neublastin in FIG. 14.

ATSSSTbSSASSASSSSSSATSTSSTSSTSSTbSSASSASSASSASbTSSSTSTSS

TSTSSSTTSTSAASTASTSSSSSSSTSSetTSSASTSSSASSSSSASASSSTTSSSS

SSAASTASTASSTTTTSSTTTTTSTSTSASSATSTTSTSTSSTSSASSTTSTSSSS

SATSATSTATSTSTSTASSATSTSTASTASSASSASSSSSSASSASTAASASSSSSSSSSSSSSSATSTA6ASSTSTSTSTAASSTTSTSTASASSTASATAS6AASSASTAT STTTSATSSASTAASTASTASATSSASASSTASATASASTASTASSASSASSASSASSASSASSASSASSASSASSASSASSASSASSASSASSASSASSASSASSASSASSASSASSASSAS6

The nucleotide sequence encoding histidine-labeled neublastin in FIG. fifteen.

ATSSSSSATSATSATSATSATSATSATSATSATSASTSSASSSSSSATATSSSSASS

ASSASSASSAASSSTSSASSASSSSSSSATSTSSTSSTSSTSSASSASSASSASSASST

SSSTSTSTSTSSSTTSTSAASTASTSSSSSTSSSTSSASTSSSSASTSSSASAS

SOTTSSSSASSAASTASTASETTTTSTSTTTTTSTTSAOOSATSTTSTSSTSSTSSAS

STSTSSSSSSATSALSTATSTSTASSATSTSTASTASSASSASSSSASSASTAASASS ASSSSSSSSSSATSTASSTSTSTSTAASSTTETTSTASASSTASTASATASSAA 6САТАТСТТТСАТССАСТААСТАСТАТАТСАССАССАТАССАСТАСТАСАТАСТАСТАСАТАСТАСТАСТАСАТАСТАСТАСАТАСТАСТАСАТАСТАСТАСТАСТАСТАТАСТАСТА

Example 6. The effect of neublastin on the survival of dopaminergic neurons in rat embryos and HAT activity.

In this series of experiments, the effect of the conditioned medium on the neublastin-producing H | B5bLP / XBX22 cells described above was evaluated.

Preparation of crops. The ventral part of the middle cerebral bladder or spinal cord was removed from opened E14 rat embryos and placed in Hanks' cold buffered saline (BSCX). Pieces of tissue were incubated in sterile filtered 0.1% trypsin (LUoPitDuy) and 0.05% DNase (81 dtA) in BSCX at 37 ° C for 20 min. Then the tissue pieces were washed four times in BSCX + 0.05% DNase and dissociated using an automatic 1 ml pipette. Then the suspension was centrifuged at 600 rpm for 5 minutes and the pellet was resuspended in conditioned medium with serum (KCC; OMEM with 10% fetal calf serum). The total number of cells was evaluated using the trypan blue stain exclusion method, and the cells were plated at a density of 100,000 cells / cm ² in poly-L-lysine coated eight-well chambers (MSC) to assess the survival of dopaminergic neurons, or at a density of 200,000 cells / cm ² 48 well plates (Mshe) for measuring HAT activity. Cells were incubated in KSS at 5% C0 ₂ /95% 0 ₂ and 95% humidity at 37 ° C for 24-48 hours before changing to serum-free medium (SBS) with the addition of neurotrophic factors.

To assess the survival of dopaminergic neurons, the cells were left for 5 days in SBS + supplementation of trophic factors and then fixed for 5 min in 4% PFA and stained for tyrosine hydroxylase using immunohistochemistry.

To determine the HAT activity, the cells were left for 3 days in SBS and then lysed in BSCX + 0.1% Triton X-100 and immediately frozen in dry ice until the HAT activity was measured.

Adding trophic factors. The conditioned medium was collected from untransfected H1B5 control cells or from H1B5 cells producing neublastin (H | B5pri1xXBX22) or SOXE (ΗίΒ5ρυΐ> ί1ζ6 ECE-E17). Η ^ B5ρυЬ ^ 1ζNВN22 produces approximately 20 ng 00 ^ / 24 h / 10 ⁵ cells, as determined by SOXE-EKE in an conditioned medium collected from cells. Corresponding cell lines were incubated overnight in EMEM + 1% PBS and the supernatant was taken and stored at -20 ° C until use. When added to the cells, supernatants were diluted 1:50 in SBS. Separate wells were treated with an supernatant from H1B5 control cells (1:50) + purified recombinant rat SENE (from 0.03-10 ng / ml).

The results of these experiments are shown in FIG. 4. FIG. 4A-4C are illustrations of the effect of neublastin secreted by Η ^ B5ρυЬ ^ 1ζNВN22 cells on the survival of cultured dopaminergic neurons in the ventral part of the middle cerebral bladder of rat embryos and on CAT activity in cholinergic motor neurons of the cranial nerve in a serum-free medium as described below in 5.1.

FIG. 4A is an illustration of the dependence of HAT activity on the dose of recombinant SOIE (dec. / Min / h) measured at ΌΙν 5 in serum-free cultures that were originally created from the ventral part of the midbrain bubbles of the E14 embryos [i.e. HB5; SOYA 0.03 ng / ml; VOI 0.1 ng / ml; BOIE 0.3 ng / ml; SECE 1 ng / ml; SECE 10 ng / ml; SECE 100 ng / ml].

FIG. 4B is an illustration of HAT activity (dec. / Min / hr) measured on ΌΙν5 in serum-free cultures that were originally created from the ventral part of the midbrain bubbles of E14 embryos. The diluted conditioned medium was either from SHB ^^ and NSE # 22 (Neublastin) producing neublastin cells or OKOE producing <^ B56 ^ NE ^ -17 (ΟΌ№ΟΌ-17) cells, as shown in the figure [i.e. neublastin 1:10; neublastin 1:50; 6E No. E-17 1:50].

FIG. 4C is an illustration of the number of immunoreactive cells in a tyrosine hydroxylase assay in a well [N0. TG ⁺ cells / well] on ΌΙν7 in serum-free cultures that were originally created from the ventral parts of the midbrain bubbles of rat embryos E14. The diluted conditioned medium was either from untransfected H1B5 (H1B5) cells or from neublastin producing cells Η ^ B5ρυЬ ^ 1ζNВN22 (neublastin) or recombinant ный # was added in various concentrations, as shown in the figure [ie H1B5 1:10; H1B5 1:40; СО№

O, 1 ng / ml; ΟΌ No. 10 ng / ml; ΟΌ No. 100 ng / ml; and neublastin 1:40].

The conditioned medium from neublastin-transfected H1B5 cells at a 1:40 dilution significantly increased the number of TG immunoreactive cells per well compared to control (non-transfected) H1B5 cells at an equivalent and lower dilution (1:10 and 1:40) (see, for example , Fig. 4B). The increase in the number of TG immunoreactive cells is comparable to the increase observed at the maximum concentration of CO # (10 ng / ml). This suggests that neublastin secreted into the medium affects the survival of the population of dopaminergic neurons in the ventral part of the middle cerebral bladder of rat embryos. In contrast, unlike ΟΌ # secreted by transfected H1B5 cells, there was no effect of the conditioned medium from neblastin-transfected H1B5 cells on another population of neurons in the same culture on cholinergic neurons (see, for example, Fig. 4A).

Example 7. The effect of neublastin on the survival of cultures of slices of dopaminergic neurons of the ventral part of the middle brain bubble of pig embryos.

In this experiment, the result of co-cultivation of Neublastin-producing cells Η ^ B5ρυЬ ^ 1ζNВN22 and cultured sections of the ventral part of the middle brain bubbles of pig embryos was evaluated.

Preparation of crops. The ventral parts of the middle cerebral bladders (SCM) were isolated from pig embryos (E28; n = 12) under sterile conditions, 400 μl of sections were cut and placed in a cooled balanced Gasey saline solution (6–00) with glucose (6.5 mg / ml). Tissue sections were cultured using the method of contact cultures, originally developed δΐορρίηί s1 a1. [Ε.δΐορρίηί,

P. A. Visek, E. Mi11eg. 1 #igo 8C1. MeBuyk. 1991, 37, 173-182].

Briefly, sections placed on semipermeable membranes (M1 Schroe, 0.3 μm; 8 sections / membrane, corresponding to one SCM), were placed as inserts in 6-well plates (Soiag) in a medium containing serum (C1bco BJb). Each well contained 1 ml of medium (50% Orytet, 25% horse serum, 25% Hanks balanced salt solution (all C1Co reagents)) with the addition of β glucose to a final concentration of 25 mM. On day 0, 7000 transfected H1B5p cells υόί1ζΝΕΝ22 (neublastin) or 7000 non-transfected H1B5 cells (control) were seeded on each tissue section. Co-cultures were first grown in an incubator at 33 ° C for 48 h, which allowed H1B5 cells to become immortalized due to the temperature-sensitive oncogen so that they could proliferate, and then placed in an incubator at 37 ° C, where H1B5 cells differentiated. Wednesday was changed twice a week. Antimitotic agents and antibiotics were not used at any stage.

Determination of dopamine by HPLC. On the 12th and 21st day of cultivation, ν νίΙΐΌ, the culture medium was collected and dopamine was analyzed using HPLC with electrochemical detection (Α.Ν. 81oo111, GV.R. Sgatkegdep, 1 # 8GEM8! 1995, 60, 141-149).

Tissue processing and immunohistochemistry. On day 21, cultures were fixed in 4% paraformaldehyde in phosphate buffer for 60 minutes, dehydrated in 20% sucrose solution for 24 hours, frozen, 20 μm sections were cut (4 series) in a cryostat, and preparations were prepared on gelatin-coated glass coverslips for a microscope. One series of sections was immunostained for tyrosine hydroxylase (TG). Briefly, the sections were washed in 0.05 M Tris-buffered saline (TBS, pH 7.4) containing 1% Triton X100 for 3 x 15 min and incubated with 10% purified bovine serum (OBS, Bn TesNpo1o §1e5) in TBS for 30 minutes The tissue was then incubated for 24 hours at 4 ° C with mouse monoclonal anti-TG antibodies (VoeNppdeg MappNepp) at a dilution of 1: 600 in TBS with 10% OBS. After washing in fuel assemblies with 1% Triton X-100 for 3 x 15 min, the sections were incubated for 60 min with biotinylated antibodies against mouse 1dC (ATEGKAT) at a dilution of 1: 200 in TBS with 10% OBS. Then, the sections were washed in TBS with 1% Triton X-100 (3 x 15 min) and incubated for 60 min with streptavidin peroxidase (Paco) at a dilution of 1: 200 in TBS with 10% OBS. After washing in TBS (3 x 15 min), bound antibodies were detected by treatment with 0.05% 3,3-diaminobenzidine (81 dtA) in TBS containing 0.01% H ₂ O ₂ . At the end, the sections were dehydrated in alcohol, purified in xylene and covered with a coverslip in Ec1.

Cell counting and morphometric analysis. The number of immunoreactive TG-ir neurons was determined using bright field microscopy (O1utric). Only intensely stained cells with a well-preserved cell structure and a distinct nucleus were counted. The determination was based on cell counts in every fourth cultured section using an x20 lens. The number of cells was refined by double counting in accordance with the formula Aegotsgotie (M. Aegotsgotie, ApaEees., 1946, 94, 239-47), using the average diameter of the nuclei in TG ir neurons (6.6 ± 0.2 μm, n = 30) . The size of the nuclei was estimated using a system of labeling of neurons (No. IgO1is1ba, M1sgoVr1dY-P1e1b, 1ps.).

The results of these experiments are shown in FIG. 5. FIG. 5A-5C are illustrations of the effect of neublastin secreted from H1B5pL2No2 cells on the functioning and survival of cultured sections of dopaminergic neurons in the ventral part of the middle cerebral bladder of pig embryos cultivated either together with H1B5p cells υΜ1ζΝβΝ22 (control cells), or as described below. FIG. 5A and FIG. 5B illustrate dopamine released on Wednesday at Όΐν12 [dopamine (pmol / ml) - day 12] and Όΐν21 [dopamine (pmol / ml) - day 21], respectively. FIG. 5C is an illustration of the number of immunoreactive cells in a tyrosine hydroxylase assay in culture [TG-ir cells per culture] in Όΐν21.

HPLC analysis on day 12 showed that the medium of the joint H1B5-neublastin cultures contains 84% more dopamine than the medium of the joint H1B5-C control cultures (Fig. 5A). On day 21, the difference was 78% (Fig. 5B), and the number of cells showed that joint H1B5-neublastin cultures contained 66% more immunoreactive to tyrosine hydroxylase neurons than joint H1B5C control cultures (P <0.05) (Fig. 5B). 5C). This suggests that neublastin secreted from the H1B5pLBN2 No. clone has an effective effect on the survival of dopaminergic neurons in pig embryos.

Example 8. The survival of the cells of the ganglion of the dorsal root in serum-free medium.

This example shows the neurotrophic activity of a neublastin polypeptide in comparison with known neurotrophic factors.

Pregnant female mice were sacrificed by displacement of the cervical vertebrae. The embryo was cultured as follows.

Electrolytically pointed tungsten needles were used to dissect the ganglia of the dorsal roots at the indicated stages in C57 / B16 mice (Mo11edaagb Vgeebtd, Oeptagk). Embryonic ganglia were incubated for 5 min at 37 ° C with 0.05% trypsin (C1bCO / WKB) in Hanks balanced salt solution without calcium and magnesium. Postnatal ganglia were treated with 1 mg / ml collagenase / dispase for 30 to 45 minutes and then with trypsin / DNase 0.25% for 15 minutes. After removal of the trypsin solution, the ganglions were washed once with 10 ml of BMEM containing 10% heat-inactivated horse serum and carefully triturated with a Pasteur pipette with a melted end to obtain a suspension of individual cells.

Cells were plated on 24 well Schips plates), which were precoated with polyornithine (0.5 mg / ml, overnight) and laminin (20 mg / ml for 4 hours; Clb / WKB). Neurons were incubated at 37 ° C in a humidified incubator and at 5% CO2 in a specific medium containing Natz P14 supplemented with 2 mM glutamine, 0.35% bovine serum albumin, 60 ng / ml progesterone, 16 mg / ml putrescine, 400 ng / ml B-thyroxine, 38 ng / ml sodium selenite, 340 ng / ml triiodothyronine, 60 mg / ml penicillin and 100 mg / ml streptomycin.

After 48 hours of incubation, neurons were clearly distinguishable by their bipolar morphology using phase contrast optics. The percentage of neuronal survival in the absence or in the presence of trophic factors (added to the culture medium before seeding of neurons at a concentration of 10 ng / ml) or conditioned medium from neublastin-producing H1B5pb1k№№2 cells was estimated by counting neurons in the wells for 48 hours of incubation.

The results of these experiments are shown in FIG. 9, in this figure means a control experiment (in the absence of factors);

means experiments in the presence of SBF;

means experiments in the presence of neurturin;

means experiments in the presence of the neublastin of the invention;

E12 presents data from experiments performed on GDK cells (dorsal root ganglia) isolated on day 12 of embryonic development;

E16 presents data from experiments performed on GDK cells isolated on day 16 of embryonic development;

P0 represents data from experiments performed on HDC cells isolated on a birthday;

P7 presents data from experiments performed on HDC cells isolated on day 7 after birth; and

P15 presents data from experiments performed on HDC cells isolated on day 15 after birth.

These results clearly show that the neurotrophic factor of the invention exhibits activity comparable to, or even better than, the activity of well-established neurotrophic factors.

Example 9. The effect of neublastin on dopamine neurons of the black substance ίη νίνο.

To evaluate the ability of neublastin (neublastin) to protect dopamine (IA) neurons of the adult black substance from 6-hydroxydopamine-induced degeneration, the authors used a model of Parkinson’s disease in rats (8aeg apb 0eg1e1, no. 8c1epse, 1994, 59, 401-415) and transfer neublastin gene using lentiviruses.

Production of lentiviruses: to create a lentiviral gene transfer vector encoding neublastin, rNA'-neublastin, a WatH1 fragment of 1331 bp neublastin cDNA subcloned into the WatH1 / Bd1 II site of p8B301 (1 Puygodep). A WatH1 / X1o1 fragment of 1519 bp length was cut from this design. and embedded in BatH1 / X1o1 the site of the post-translational fragment of the hepatitis virus of the North American groundhog carrying the RNA '(Kwon, K. Bopen11. Lew ge1gogy1 gesUgz; ΌνπΌ1., 1999, 73 (4) 2886-2892). In order to create an rNSSBJ of the WatH1 / X1u1 fragment from rbbx-OBZh with a length of 701 bp, an rNA 'site was inserted into the BatH1 / X1o1.

Obtaining a lentiviral vector is described, for example, 2HGhegeu e! a1. (^ iGGegeu K., No.scu Ό., Mapbe1 K..B, No. 11byi B., Tgopo Ό .: MiShr1u ayepia! eb 1ep! Mga1 wes! 1997, 15 (9), 871-875). Briefly, the transfer constructs and helper plasmids pK8.91 and rMBS were jointly transfected into 293T cells. Virions released on Wednesday 48 and 72 hours after transfection were collected. In order to concentrate the virus, the medium was centrifuged for 1.5 hours at 141,000 d, and the precipitate was dissolved in BMEM. The titer of the control carrying the green fluorescence (CEP) protein gene was determined using CEP fluorescence of 293T cells and was 10 ⁸ transformation units (ET) / ml. RNA slot-blot technology (gop 8c11 \ ueb1eg i., 8opd I., A1kep S., Tgopo B.: \ | G B sis1a1 Gog yitap 1tipobeyaepsu uiiz 1ure 1 rgouia1 ^ NΑ zuShe818 1n tGes1eb se11z; ΐ Ό, 1993, ), 49454955) was used to determine the titer of viral particles. The CO # supernatant and the neublastin supernatant had 10 times fewer particles compared to the SER supernatant.

Surgical procedures: all animal work was carried out in accordance with the rules established by the Land University Laboratory Animal Ethics Committee.

We used young adult female 8gadie-0aMeu rats (B & K ishuegka1, 81osk11o1t, 8 \\ 'ebep), a total of 21 individuals, and placed them in a 12-hour light cycle: darkness with free access to food and water. Retrograde labeling and 6-0ΗΌΛ lesions were performed 3 weeks before the lesion, according to 8aeg apb 0eg1e1 (8gig apb 0eg1e1, No. 1xeps, 1994, 59, 401-415). Briefly, under the anesthesia of Edixhex (0.3 ml / 100 g), the rats were injected bilaterally with 0.2 μl of a 2% solution (dissolved in 0.9% No. 1C1) of the retrograde label E1iogo-Oo1b (EO; E1iogosgote, 1ps., Epd1e ^ SOB, C0). Injections were made using a Hamilton syringe with a volume of 2 μl to the points having coordinates: AP = + 0.5 mm; M = ± 3.4 mm relative to bregma; Όν = -5.0 mm relative to the dura mater and the root of the incisor was set to 0.0 mm. In addition, 0.05 μl / min was injected for an additional 5 minutes remaining until the needle was withdrawn.

Fourteen days after EO injections, the animals received a total of 5 injections (1 μl / injection) of a lentiviral vector carrying the green fluorescence protein gene (OEP), neublastin or 6ΌΝΕ. Four introductions were into the striatum in two directions of the needle with the following coordinates:

AP = + 1.0 mm, Mb = -2.6 mm, Όν ₁ = -5.0 mm, Όν ₂ = -4.5 mm and AP = 0.0 mm, M = -3.7 mm, Όν ₁ = -5.0 mm, Όν ₂ = -4.5 mm. The introduction of the black substance was done at AP = -5.2 mm, Mb = -2.0 mm, Όν ₁ = -6.3 mm. The root of the tooth was achieved at -2.3 mm.

Twenty-one days after retrograde labeling and 7 days after lentivirus injections, the animals were re-anesthetized and a single dose of 20 μg 6-0NEA was injected with a 10 μl Hamilton syringe (8 pcs; calculated as the free base and dissolved in 3 μl of ice-cold salt solution with addition 0.02% ascorbic acid) in the right striatum in the same place as the introduction of EO. The injection rate was 1 μl / min, and was left for another 3 minutes until the needle was removed.

Tissue treatment: on day 21 after 6-0 NEA injection, the animals were deeply anesthetized with chloral hydrate and perfused through the heart with brine (pH 7.4; room temperature) for one minute, followed by 200 ml of ice-cold formaldehyde solution (4% paraformaldehyde at 0 , 1M phosphate buffer, pH 7.4). The brain was dissected and re-fixed in the same fixative for 3-4 hours and then transferred to 25% sucrose / 0.1 M phosphate buffer for 48 hours. Five series of 40 μm sections of the striatum and black substance (CV) were cut using a freezing microtome.

Quantification of dopaminergic neurons in FV: the number of EO-labeled neurons in the CGI spermatida vaginalis was evaluated by blind observation, as described previously (8aeg apb 0g1e1, No. hydroxysepse, 1994, 59, 401-415). Briefly, three consecutive sections were used at approximately the level of the medial terminal nucleus of the accessory optic tract (MTN; -5.3 in the atlas Rakhshok apb \ Wa1kop (1997)), and all labeled / stained neurons located laterally with respect to MTN were counted at 40x magnification (n = 6-7 / group). EO-labeled neurons were included in the count if they brightly fluoresced under epi-illumination at 330 nm, the parameters of neurons were detected, and their participation was extended to at least one neuritic process.

On the injured side in animals receiving injections of lentiviruses bearing OEP, the number of EO-positive neurons of the black substance was reduced to 18% of the amount on the intact side. In contrast, in animals injected with lenti-neublastin, almost complete protection of the number of EO-positive black matter neurons was observed (89%). It was also effective, as in animals treated with Lenti-OP #, where 87% of retrogradely labeled neurons remained on the injured side. This suggests that neublastin is an effective factor in ensuring the survival of damaged dopaminergic neurons of the black substance of an adult organism, and that it is as effective as βΌΝΓ.

FIG. 6 is an illustration of the effect of neublastin produced by lenti viruses on dopamine neurons of the black substance w U1yo. The neurons of the papilla sotras! And the FV in female rats 8gadie Oa \\ '1eu were retrogradely labeled E1igodo1b (EO) 3 weeks before a single injection of 6-hydroxydopamine (6-0NOA) in the right striatum. The week before the injection of 60 NEA, the animals received injections of lentiviral vectors expressing neublastin [neublastin], OO№ [OO№], or green fluorescence protein [OEP], as shown in the figure. Twenty-one days after 6-0 NEA injections, the number of E-labeled striatum neurons on both sides was determined. The figure shows the percentage of [% EO damaged / intact] EO-labeled neurons in the striatum on the damaged (right) side compared with the striatum on the intact (left) side in three groups of animals.

Example 10. Obtaining antibodies.

To prepare antibodies against neublastin, two rabbits at 3 week intervals were immunized with either peptide 1: САРТΒΥΕВ8ЕМ ^ VN8Т (amino acids 108124 in 8ЕО ΙΌ Ν0: 9); either peptide 2:

ABBRRSBBRUBrRS (amino acids 93-107 in BEC ΙΌ N0: 9) conjugated to a protein carrier. Two rabbits for each peptide were immunized at 0, 3, 6, and 10 weeks, and blood samples were collected at 7 and 11 weeks. Second blood samples were affinity purified on affinity protein columns. Antibodies were named At-1 and At-2, according to the peptides.

Western blot: 2 x 10 ⁶ H1B5 cells stably transfected with neublastin cDNA (H ^ L5puB ^ 1ζNBN22) or untransfected H1B5 cells were incubated overnight in serum-free medium supplemented with N (C1BC0). The medium was concentrated on small concentrators with cut membranes at 5 kD (M1Shroge, WebGogb, MA). 5 x Laemmli sample buffer was added to the concentrated samples and heated to 95 ° C for 5 minutes. Samples were separated by electrophoresis in a BOB polyacrylamide gel in 15% acrylamide gels and transferred to RUER membranes. The remaining protein-binding sites were blocked with a 5% solution of skimmed milk powder in FSB with 0.1% tween-20. Membranes were incubated overnight with antibodies against neublastin (1: 1000), after which they were incubated with second antibodies against rabbit or mouse 1dC conjugated to horseradish peroxidase (1: 2000).

Immunostaining was visualized using enhanced chemiluminescence plus (UHL +) in accordance with the manufacturer's instructions (ATEGKAT). The results of these experiments are shown in FIG. 3 and in example 5.

Using standard methods, the authors also obtained rabbit polyclonal antibodies against the following peptides:

peptide B27: SRSBVAWAASAVSS (amino acids 30-43 BEC ΙΌ N0: 9);

peptide B28: LCHNBPHEHWRBRS (amino acids 57-70 BEC ΙΌ N0: 9);

peptide B29: ATTENTION (amino acids 74-85 BEC ΙΌ N0: 9);

peptide B30: EVRRRSBVRUBSRS (amino acids 94-107 BEC ΙΌ N0: 9);

peptide B31: BT ^ VTUEKBATAS (amino acids 123-136 BEC ΙΌ N0: 9).

Only peptides B30 and B31, located relatively close to the C-terminus, recognized the denatured protein under reducing conditions on a Western blot.

<160> 33 <170> Patent Version 2.0 <210> 1 <211> 865 <212> DNA <213> Noto δδρίβπδ <220>

<221> The coding sequence <222> (120) .. (719) <220>

<221> 5'NTR <222> (1) .. (119) <220>

<221> Z'NTR <222> (721) .. (865) <220>

<221> signal peptide <222> (120) .. (179) <220>

<221> mature peptide <222> (405) .. (719) <220>

<221> tbcc structure <222> (661) .. (663) <223> CARBOGID: Glycosylated asparagine at Azp87 position <220>

<221> TCS structure <222> (426) .. (623) <223> DISULFIDE - Suz8-Suz73 disulfide bridge <220>

<221> T1ZS ~ structure <222> (507) .. (707) <223> DISULFIDE - Suz35-Suz101 disulfide bridge <220>

<221> tbcc structure <222> (519) .. (713) <223> DISULFIDE - Suz39-Suz103 disulfide bridge <220>

<221> shhss-structure <222> (616) .. (619) <223> DISULFIDE - Suz72-Suz72 disulfide bridge <400 1 sEddadssss addssssdesdess

asd sss mes rgo -95 dss A1a sed bay Edd sss ase sed dss DSS CED DSS CED Ede Ad Ade A1a Bei Ayia Bei Bei Zeg Zeg 167 Tgr Rgo Th2 -90 Bei Aya -85 -80 dss dsa dad dss STS ssd dds Sss grandfather sss sds hell CCE dss sss sds 215 Ya1 A1a C1 and A1a Zeg -75 Bei 31u Aya RGO -70 Ltd. Run Mouth Aya Rge -65 Agd yeah sds STS ssd STS dss garden grandfather Sss STS dss dds sas eat ssd YAYAYA 263 C1 and C1u Rgo Rgo -6C Mouth Ya1 Bei Aya Zeg -55 Rgo Aya 01y H1z Bay -50 Rgo Xiu dda sds asd dss sds SDD sas hell hell hell dss edd garden ssd sds sds 311 C1u Agd Bt -45 A1a Agd Tgr Suz Zeg -40 <31u Agd Aya Agd Agd -55 Rgo Agd Agd hell sas sss Ssd dss sds dss sss dss dss Food ace sss ace Food Ese 359 Agd H £ * -30 Pb Zeg A1a Agd A1a -25 Rgo A1a Aya Suz Bt -20 ? go Not Suz Zeg Sss ssd edd dss sde grandfather grandfather edd ssd ddd dds edd dsa des sds Ssd 4C7 Zeg -15 Rgo Agd Ya1 Agd A1a-10 Aya Agd Bei 31 / C1u Agd Aya Aya Agd Zeg one dds hell YAYAYA dds grandfather YAYAYA Vds sds sed sds Eating garden sed grandfather ssd grandfather 455 C1u Zeg C1u C1u 5 A1e 31u Suz Agd Bai 10 Agd Zeg Ch-1P Bei 7a1 fifteen RGZ Ya1 sds grandfather CEC dds ssd dss sas eating sss das dad eat grandfather cde HER: sds 503 Agd A1a Bei 23 C1u Bei C1u Ηίδ Agd Zeg Azr C1 and Bei ν & ι Agd Pb Agd ses eating ace dds Sss Vds ssd sds grandfather sds ECE ssa sas das sss hell 551 Pb Suz 35 Tht s1u Zeg Suz Rgo 40 Agd Aya Agd Zeg Rgo 45 Her" Azr Bei Zeh sed dss hell ssa garden dds dss YAYAYA dss ssd food sed sss ssd dd: Ese 599 fifty A1a Zeg Bei Bei 61y 55 A1a C1 ’/ Aya Bei Agd 60 Rgo Rgo Rgo C1u Zeg 65 edd STS dss hell garden sss sds Vds food sss asd sds EEC yeah grandfather dss 647 Agd Rgo Ya1 Zeg C1p 70 Rgo Su Suz Agd Rgo 75 Tht Agd Tug 21and Aya 80 Wye Sss With EU aU das dss aah hell ace ddd hell ace grandfather das sds CEC STS 695 Zeg Pb Mee Azr 85 Ya1 Az η Zeg Tht Tgr 90 Agd Tht ν> 1 A = p Agd 95 Bei Zeg dss A1a ace Tht dss A1a one hundred sde suz dds <31u sds souz ssd Bei RL <= C1y 105 Sddadddssss esssadddss ss ds adced 749

Sequence Listing

Mek Rgo Aya Bie Tgr Rgo YOU Bie Aya Aya Bie Aya Bie Bie Zeg Zeg -95 -90 -AT 5 -80 Wye Aya Sy Aya Zeg Bie Oyu Zeg Aya Rgo Agd Zeg Rgo Aya Rgo Agd -75 -70 -65 Sy Oyu ? gs Rgo Rgo Wye Bie Aya Zeg Mouth Aya Xiu Ηίβ Bie Rgo Oyu -60 -55 -fifty Oyu Agd Tht Aya Agd Tgr Suz Zeg Oyu Agd Aya Agd Agd Rgo Agd Agd -45 -40 -35 Agd ΕΪ3 Pb Zeg Aya Agd Aya Rgo Aya Aya Suz YOU Rgo Not Suz Zeg -thirty -25 -twenty Zeg Rgo Agd Wye Agd Aya Aya Agd Bie Xiu <31u Agd Aya Aya Agd Zeg -fifteen -10 -5 -one one Oyu Zeg Xiu Oyu Aya Xiu Suz Agd Bie Agd Z C1p Bie Wye Rgo Wye 5 10 - ⁵ Agd Aya Bie Xiu Bie Xiu H1z Agd Zeg Azr C1 and Bie Wye Agd Pb Agd twenty 25 thirty Pb Suz Tht Oyu Zeg Suz Rgo Agd Aya Agd Zeg Rgo Nhz Azr Bie Zeg 25 40 45 Bie Aya Zeg Bie Bie Oyu Aya Xiu Aya yee Agd Rgo RGO Rgo Oyu Zeg fifty 55 60 65 Agd Rgo Wye Zeg Oyp Rgo Suz Suz Agd Rgo YOU Agd Tug Sy Aya Wye 70 75 80 Zeg Pb Mek Azr Wye Azp Zeg Tht tgr Agd Tht Wye Azr Agd Bie Zeg 85 90 95 Aya Tht Aya Suz Oyu Suz Bie Oyu one hundred 105

<210> 3 <211> 861 <212> DNA <213> Noto sarheps <220>

<221> The coding sequence <222> (7) .. (717) <220>

<221> 5ΉΤΡ <222> (1) .. (6) <220>

<221> Z’NTR <222> (718) .. (861) <220>

<221> signal peptide <222> (7) .. (174) <220>

<221> mature peptide <222> (298) .. (717) <220>

<221> mature peptide <222> (370) .. (717) <220>

<221> mature peptide <222> (379) .. (717) <220>

<221> gpdcs structure <222> (661) .. (663) <223> CARBOGID: Glycosylated asparagine at position Azp122 <220>

<221> TCS structure <222> (424) .. (621) <223> DISULFIDE: Suz43-Suz108 disulfide bridge <220>

<221> tyses structure <222> (505) .. (705) <223> DISULFIDE: Suz70-Suz136 disulfide bridge <220>

<221> tyses structure <222> (517) .. (711) <223> DISULFIDE: Suz74-Suz138 disulfide bridge <220>

<221> tyzs-structure <222> (616) .. (618) <223> DISULFIDE: Suz107-Suz107 disulfide bridge between chains <400> 3 dadss asd ase ade skd als Ssa dss food dda garden sss sss ekk dad43

Mek Rgo 61y Lei Ne Zeg A1a Agd C1u S1p Mego Rgo Ei Ley S1i

-95 -90-85

duke ekk wye bie sec sss saa <31p dss sas sed ddk dss sks kkk sks sec dad dec 96 Rgo RGO -80 Ayah Nyz Bie Oyu A1a -75 Lye Rye Bie Rgo Oyi Ayia -70 ssa ekk dds SCS css grandfather garden FROM" dss csd cdd sss ace ssd dss dss 144 Bie Well -65 Bie Zeg Aya S’p RGO -60 Aya Bie Tgr Rgo YOU -55 Bie Aya Aya skd Dec with cd ssd hell hell dss dsa dad dss Sss skd dds STS grandfather sss 192 Bie Aya -50 Bie Bie Zeg Zeg Wye -45 Aya Sy Aya Zeg Ley -40 Xiu Zeg A1a Rgo sds hell sec dss sss sds yeah dds sss ssd ssk dke ssd grandfather Sss sss 240 Agd -35 Zeg Rgo A1a Rgo Agd -30 Sy Oyu Rgo Rgo Rgo -25 Wye Bie Aya Zeg RGO -20 dss dds sae with cd ssd ddd dda sds asa dss sds cdd Vds hell dda hell 288 Aya Oyu Nyz Bie Rgo -15 Xiu Xiu Agd YOU Ayah Agd Tgr Suz Zeg syu -5 Agd dss edd edd ssd ssd ssd garden ssk ssk edd sss grandfather sss ssd ssd ssk 336 Aya Agd Agd -one Rgo Rgo Rgo Syp Rgo 5 Zeg Agd Rgo A1a Rgo 10 Rgo Rgo Rgo dsa STS ssa cake Dec ssk sss sds ddd dds sds grandfather grandfather edd dss ddd 384 Aya Rgo fifteen Rgo Zeg Aya Bie Rgo twenty Agd Oyu Xiu Agd Aya 25 Aya Agd Aya Xiu dds sed dds aas sds Dec edd dsa grandfather ddd ssd edd dds sds sds skd 432 Oyu thirty Rgo <31u Azp Agd Aya 35 Agd Aya Aya Xiu Aya 40 Agd Xiu Suz Agd Bie 45 sds sneaker garden with cd dkd ssd grandfather sds grandfather SCS dds skd eating sas sds sss 480 Agd Zeg From 1g. Bie Wye fifty RGO Wye Agd Aya Bie 55 Oyu Bie Xiu Nhz Agd 60 Z das dad ssd dkd caustic cx sds cx sds hell dds css Vds sds sds grandfather 528 Azr Sy Bie Wye 65 Agd Pb Agd Ri Suz 70 Zeg Oyu Zeg Suz Agd 7C Agd Aya sds cake ssa sae das SCS hell ssd dss hell ska skd dds dss ddd dss 576 Agd Zeg Rgo in Ηίε Azr Bie Zeg Bie 35 Aya Z Bie Bie Xiu 90 Aya Xiu Aya cζд food ssd sss ssd dds Sss edd sss dss hell garden sss Vds sds food 624 Bie Agd 95 Mouth Rgo Rgo C1u Zeg one hundred Agd Rgo Wah! Zeg Syp 105 Rgo Suz Suz Agd STS asd sds to ac yeah grandfather dss sss css asd das dke aas hell ace edd 672 ? go 110 YOU Agd Tug Sy Aya 115 Wye Zeg Pb Month Azr 120 Wye Azp Zeg YOU Tgr 125 Ada Agde ace YOU dkd wye das azr sds agd 130 sks bie css Zeg dss aya aas Azp sss rgo 135 kde soz dds syu SDS Souz skd lei dds oyu 140 717

cdddddsscd skssadddsk ckdsadascd dasskkass dddcdsksk sskdsdcdd 777 asssssssdsd adadkssas Sadssadssd ssksadssad ddddaadsd sksaaadssd 837

Mek Rgo syu Bie Not Zeg Aya Agd Yo Syp Rgo Bie Bie Sy Wye Bie -95 -90 -85 Rgo Rgo Syp Aya H1z Bie Oyu Aya Bie Pb Bie Rgo Sy Aya Rgo Bie -80 -75 -70 Oyu Bie Zeg Aya ip Rgo Aya Bie Tgr Rgo YOU Bie Aya Aya Bie Aya -65 -60 -55 -5C Bie Bie Zeg Zeg Wye Aya Sy Aya Zeg Bie Oyu Zeg Aya Rgo Agd Zeg -45 -40 -35 Rgo BUT! but Rgo Agd Sy Xiu RTZ Rgo Rgo Wye Bie Aya Zeg Rgo Aya oh -thirty -25 -twenty Nyz Bie Rgo Xiu Xiu Agd YOU Aya Agd Tgr Suz Zeg Xiu Agd Aya Agd -fifteen -10 -5 Agd Rgo Rgo Rgo Syp. Mouth Zeg Agd Rgo Aya Rgo Rgo Rgo Rgo Aya Rgo - ^{1 5} 10 fifteen Rgo Zeg Aya Bie Rgo Agd Oyu 31u Agd Aya Aya Agd Aya Xiu Xiu Rgo twenty 25 AP Oyu Azp Agd Aya Agd A1a Aya oh A1a Agd Xiu Suz Agd Bie Agd Zeg 35 40 45 Syp Bie Wye Rgo Wye Agd Aya Bie Oyu Bie Xiu Nyz Agd Zeg Azr Sy fifty 55 60 Bie Wye Agd Pb Agd Pb Suz Z Oyu Zeg Suz Agd Agd Aya Agd Zeg 65 70 75 Rgo ΕΪ3 Azr Bie Z Bie Aya Zeg Bie Bie Xiu Aya Xiu Aya Bie Agd 80 85 90 95 Rgo Rgo Rgo Xiu Zeg Agd Rgo Wye Zeg Oyp Rgo Suz Suz Agd Rgo YOU one hundred 105 110 Agd Tug Sy Aya Ya1 Zeg Pb Mek Azr Wye AZP Zag YOU Tgo Agd Tht 115 120 125 Wye Azr Agd Bie Zeg Aya Azp Rgo Suz Oyu Sub Bie Xiu 130 135 140

<210> 5 <211> 140 <212> protein <213> Noto zarhepz <220>

<223> where Haa at position 134 means Azp or Tb, and ¥ aa at position 135 means A1a or Prgo <400> 5

Rgo Rgo Rgo C1p Rgo Zeg Agd Rgo A1a Rgo Rgo Rgo Rgo Aha Rgo RGO one 5 10 fifteen Zeg Aha Bie Mouth Agd C1u C1u Agd Aha Aha Agd Aha SHU Zhu Rgo boo twenty 25 thirty Azp Agd A1a Ahd Aha A1a <31u A1a Ahd 31u Suz Agd Bie Agd Zeg C1p 35 40 45 Bie Cha1 Rgo CHAH Agd Aha Bie SHU Bie SHU ΗΪ3 Agd Zeg Ayar SHi Bie fifty 35 60 Cha1 Agd Pb Agd Pb Suz Z C1u Zeg Suz Agd Agd A1a Agd Z RGO 65 70 75 80 H15 Azr Bie Zeg Bie Aha Zeg Bie Bie <51y A1a C1u Aha Bie Agd Rgo 85 90 95 Rgo Rgo <Zhu Zeg Agd Rgo Cha1 Zeg C1p Rgo Suz Suz Agd Rgo Tht Agd South Ossetia 105 110 Tug Zhi A1a Cha1 Zeg Pb Not c Azr Cha1 Azp Zeg T. bg Tgr Agd Tht Cha1 115 120 125

Azr Agd Ley Zeg A1a Haa Waa Suz <5Χν Suz Lye Zhu 130 135 140

<210> b <211> 116 <212> protein <213> Noto zarhepz <220>

<223> where Haa at position 110 means Azp or Tb, and Waa at position 111 means A1a or Prgo

<400> 6 A1a Aha Agd A1a C1u b1u Rgo C1u Azp Agd A1a Agd A1a Aha SHU Aha one 5 10 fifteen Agd C1u Suz Agd yee Agd Zeh 31p le CHAH RGO Cha1 Agd Aha Bie C1u twenty 25 thirty Bie <31u H1z Ahd 5eg Azr C1 and Ley Wa1 Agd Pb Ahd Pb Suya Zeg SHU 35 40 45

Zeg Suz Agd Agd A1a Agd Zeg Rgo N1z Azr Ley Zeg Ley A1a Zeg Ley

5560

Ley C1u A1a C1u A1a Ley Agd Rgo Rgo Rgo C1u Zeg Agd Rgs Wa1 Zeg

70 7580

С1п РГс Суз Суз Агд Рх ТЬг Agд Туг <51 and A1а Ча1 Зег РЬе МеС Азр 05 9095

Cha1 Azp Zeg Thg Thr Agd Thg Cha1 Azr Ahd Ley Zeg A1a Haa Waa Suz

US 105110

C1u Suz Ley C1u

115 <210> 7 <211> 113 <212> protein <213> Noto sarheps <220>

<223> where Haa at position 107 means Azp or Tb, and Waa at position 108 means AXa or Prgo <400> 7

A1a C1u one <31u Rgo C1u Azp 5 Agd A1a Agd A1a AXa CXu A1a 10 Aga Xu Suz fifteen Agd Bie Agd Zeg C1p Bie Cha1 Rgo Cha1 Agd A1a Bie C1u Bie SHU Ηί3 twenty 25 thirty Ahd Zeg Azr (31and Bie Cha1 Agd Pb Agd Pb Suz Zeg SHU Zeg • Souz Agd 35 40 45 Agd Aha Agd Zeg Rgo I1z Azr Bie Z Bie A1a Zeg Bie Bie SHU A1a fifty 55 60 C1u A1a Bie Agd Rgo Rgo Rgo O1u Zeg Agd Rgo Cha1 Zeg Zhp RGS Suz 65 70 75 80 Suz Agd RGS Tht Agd Here SHi A1a cha! Veg Pb MeC Azr CHAH AZP Zeg 85 90 95 Tht Tgr Agd Tht Cha1 Azr Agd Bie Zeg A1a Haa Yeah Suz C Χν Suz Bie one hundred 105 xx'o <51y <210> 8 <211> 861 <212> DNA <213> Noto zarhepa

<220>

<221> The coding sequence <222> (58) .. (717) <220>

<221> 5ΉΤΡ <222> (1) .. (57) <220>

<221> Z'NTR <222> (718) .. (861) <220>

<221> signal peptide <222> (58). . (174) <220>

<221> mature peptide <222> (298) .. (717) <220>

<221> mature peptide <222> (370) .. (717) <220>

<221> mature peptide <222> (379) .. (717) <220>

<221> thcc structure <222> (661) .. (663) <223> CARBOGID: Glycosylated asparagine at position Azp122 <220>

<221> TGHS structure <222> (424) .. (621) <223> DISULFIDE: Suz43-Suz108 disulfide bridge <220>

<221> t1cc structure <222> (505) .. (705) <223> DISULFIDE: Suz70-Suz136 disulfide bridge <220>

<221> shhss-structure <222> (517) .. (711) <223> DISULFIDE: Suz74-Suz138 disulfide bridge <220>

<221> TCS structure <222> (616) .. (618) <223> DISULPHIDE: Suz107-Suz107 disulfide bridge between the chains <400> 8 addddddsdd dddaasadse saasaSdds sdaDdddssds dessda ddssda ddssda57 Sss sas sds sss sdd 105

MeS C1i Ley C1u Ley C1u C1u Ley Zeg Thr Thy Zeg Nhz Su in RgoTgr

-80 -75 -70-65

sse add sdd garden ss dss rgo agd agd shp rgo a1a ssd sdd sss ase esd dss dss ssd dss sed aha lei -50 153 Bie Tgr Rgo Thr -55 Bie A1a aha lea -60 cd hell hell dss dsa dad dss Sss ssd dds Sss grandfather sss sds hell ssS 201 Bie Zeg Zeg Cha1 Aha SHi Aha Zeg Bie C1u Z Aha Rgo Agd Zeg RGO -45 -40 -35 dss sss sds yeah dds sss ssd sss dss csd grandfather Sss sss Yass dds sas 249 A1a Rgo Agd O1i C1u Rgo Rgo Rgo Cha1 Bie Aha Zeg RGO Aha C1u NHC -thirty -25 -twenty csd ssd ddd dda sds asd dss sds SDD eating hell dda hell dss sdd sdd 297 Bie Rgo SHU C1u Agd Tht Aha Agd Tgr = Y5 Z SHU Ahd Aha Agd Agd -fifteen -10 uh -one ssd ssd ssd garden ssS Sss sdd sss grandfather STS ssd ssd ess dsa STS ssa 345 Rgo Rgo Rgo C1p Rgo Zeh Agd Rgo A1a RGS Rgo Rgo RGS Aha Rgo Mouth ^one 5 10 fifteen sss dss ess sss eating ddd dds sds grandfather grandfather sdd dss dd? dds ssd dds 393 Zeg A1 & Bie Rgo Agd SHU SHU Agd Aha A1a Agd Aha SHU SHU Rgo SHU twenty 25 thirty hell sds dss sdd dsa grandfather ddd grandfather sdd dds sde sds sed sde ssd garden 441 Zeg Agd A1a Ahd Aha Aha C1u Aha Agd C1u Suz Agd Bie Agd Zeg SHP 35 40 45 csd dsd ssd dsd sds grandfather eСс dds csd dds sas sds Bss das dad ssd 489 Bie CHAH Rgo CHAH Ahd A1a Bie SHU Bie C1u H1z Ahd Zeg Azr C1 and Bie fifty 55 60 d ^ d sds STS sds SSc sds hell dds STS sds sds sds grandfather sds ess ssa 537 Cha1 Agd Pb Agd Pb Suz Zeg 81y Zeg Suz Agd Agd A1a Agd Zeg Rgo 65 70 75 80 sas das ssc hell eSd dss hell ssa sed dds dss ddd dss led food ssd 585 ΗΪ3 Azr Bie Zeg Bie A1a Zeg Bie Bie C1u A1a (51u Aha Bie Agd Rgo 85 90 95 sss ssd dds STS sdd sss dss hell garden STS Vds eating food sss asd sds 633 RGS Rgo C1u Zeg Agd Rgo CHAH Zeg C1p Rgo Suz Suz Agd Rgo Tht Agd one hundred 105 110 Sas yeah grandfather dss STS STS ASD das dss aas hell ace idd hell ace grandfather 681 tug C1 and A1a Cha1 Zeg Pb MeC Azr Cha1 Azp Zeg Tht Tgr Agd Tht Cha1 115 120 125 das sds sss Sss dss ace dss Vds dds sds ssd dds Sdddddss ; sd 727 Azr Agd Bie Zeg A1a Tht A1a Sug C1u Suz Bie C1u

130135140 sSssadddsS sSdsadasd dasssSSass ddSddsSsSS ssSdssSddd asssSsssds 787 adadssssas Sadssadsdd ssSsadsead ddasdaadds sSsaaadsSd adaddssssS 847 assddsddds dasd ³⁶¹

<210> nine <211> 220 <212> protein <213> But that ; <4E0> nine

MeC <31 and Ley C1u Ley C1u C1 y Zeg Tg Ley Zeg Bottom -73 Suz Rgo Tgr -65 -80 -75 Rgo Agd Agd C1p Rgo A1a Bie Tgr Rgo Tag Bie A1a Hell a Bie A1a Bie -60 -55 -fifty Bie Zeg Zeg Ya1 A1a C1 and A1a Zeg Bie • 31u Zeg A1a Rgo Agd Zeg Rgo -45 -40 -35 A1a Rgo Agd C1 and S-1u Rgo RGS Rgo 7a1 Bie A1a Zeg Rgo A1a <31u ΗΪ5 -thirty -25 -twenty Bie Rgo C1u C1u Ahd Tag A1a Agd Tgr Suya Zeg C1u Agd A1a Agd Agd -fifteen -10 -5 Rgo Rgo Rgo C1p Rgo Zeg Agd Rgo A1a Rgo Rgo Rgo Rgo A1a Rgo Rgo one 5 10 fifteen Zeg A1a Bie Rgo Agd b! y <31y Agd A1a A1a Agd A1a 51u 31u Rgo C1u twenty 25 thirty 5et Agd A1a Agd A1a A1a (31u A1a Agd C1u Suz Agd Bie Agd Zeg C1p 35 40 45 Bie ν »ι Rgo Ya1 Agd A1a Bie <31u Bie b1u ΗΪ3 Agd Zeg Azr C1 and Bie fifty 55 60 V "! Agd Pb Agd Pb Suz Zeg C1u Zeg Suz Agd Agd A1a Agd Zeg Rgo 65 70 75 80 ΗΪ5 Azr Bie Zeg Bie A1a Zeg Bie Bie C1u A1a C1u A1a Bie Agd Rgo AT 5 90 95 Rgo Rgo C1u 5eg Agd Rho Ya1 Zeg C1p Rge Suz Suz Agd Rgo Tht Agd one hundred 105 110 Tug O1i A1a 7a1 Zeg RNe Month Azr Ya1 Azp Zeg Tht Tgr Agd Tht Ya1 115 120 125 Azr Agd Bie Zeg A1a TNG A1a Suz O1u Suz Bie C1u 130 135 140

<210> 10 <211> 140 <212> protein <213> Noto zarhepz <220>

<221> CARBOGYDE <222> (122) <223> glycosylated asparagine <400> 1C

Rgo one Rgo Rgo C1p Rge Zeg Agd Rgo A1a Rgo Rgo Rgo Rgo Rgo A1a Rgo Rgo 5 10 fifteen Zeg A1a Bie Rgo Agd C1u C1u Agd A1a A1a Agd A1a C1u C1u Rgo 01 y twenty 25 thirty Zeg Agd A1a Agd Hell a A1a C1u A1a Agd O1u Suz Agd Bie Agd Zeg C1p 35 40 45 Bie Ya1 Rgo ν »ι Agd A1a Bie (51u Bie C1u ΗΪ5 Agd Zeg Azr 31and Bie fifty 55 6C Ya1 Agd Rie Agd Pb Suz Zeg 61y Zeg Suz Agd Agd A1a Agd Zeg Rgo 65 70 75 80 ΗΪ5 Azr Bie Zeg Bie A1a Zeg Bie Bie C1u A1a C1u A1a Bie Agd Rgo 85 90 95 Rgo Rgo C1u Zeg Agd RGS νβΐ Zeg C1p RGS Suz Suz Agd RGS Tht Agd one hundred 105 110 Tug 31and A1a 7a1 Zeg Pb MeC Azr 7a1 Alp. Zeg Tht Tgr Agd Tht Ya1 115 120 125 Azr Agd yee Zeg A1a Tht A1a suz C1u Suz Bie -31u

.30 135 140 <210> 11 <211> 116 <212> protein <213> Noto sarheps <220>

<221> CARBOGID <222> (98) <223> glycosylated asparagine

<400> 11 A1a A1a Agd A1a C1u & 1u Rgo C1u Zeg Ahd A1a Agd A1a A1a e1u A1a ^one 5 10 fifteen Agd C1u Suz Agd Bie Agd Zeg C1p Bie νβΐ Rgo Ya1 Agd A1a Bie 31u twenty 25 thirty Bie C1u Ηίβ Agd Zeg Azr C1 and Bie Ya1 Agd Pb Agd Pb Suz Zeg <31 y 35 40 45 Zeg Suz Agd Agd A1a Agd Zeg Rgo ΗΧΞ Azr Bie Zeg Bie A1a Zeg Bie fifty 55 60 Bie C1u A1a C1u A1a Bie Agd Rgo Rgo Mouth S-1u Zeg Agd Rgo 7a1 Zeg 65 70 75 80

C1p Rgo suz Suz Agd Rgo Tht Agd Tug 31and A1a Wa1 Zeg Rye MeC Azr 85 90 95 Ya1 AZP Zeg Tht Tgr Agd Tht Ya1 Azr Agde Lee Zeg A1a TNG A1 & Suz one hundred 105 110 SPU Suz Bie 61y 115

<210> 12 <211> 113 <212> protein <213> Noto zargetz <220>

<221> CARBOGID <222> (95) <223> glycosylated asparagine <400> 12

A1a C1u C1u Rge C1u Zeh Agd A1a Agd A1a A1a C1u A1a Ahd e1u Suz one 5 10 fifteen Agd Bie Agd Zeg <E1p Bie νβΐ Rgo Ya1 Agd A1a Bie C1u Bie C1u H1z twenty 25 thirty Ahd Zeg Azr 31and Bie Ya1 Agd Pb Ahd Pb Suz Zeh O1u Zeg Suz Ahd 35 40 45 Agd Hell a Agd Zeg Rgo Nhz Azr Bie Zeg Bie A1a Zeh Bie Bie C1u A1a fifty 55 60 61y A1a Bie Ahd Rgo Rgo Rgo C1u 5eg Ahd Rgo νβΐ Zeg <31p Rho Suz 65 70 75 IN Suz Agd Rgo Bx Ahd Tukh <3x A1a Ya1 Zeg Pb Month Azr Ya1 Azp Z 85 90 95 Bx Thr Ahd Tht Ya1 Azr Ahd Bie Zeg A1a Bx A1a Suz C1u Suz Bie one hundred 105 110

C1u <210> 13 <211> 102 <212> DNA <213> Notarheps <400> 13 ssdddssads sEasDddsd ssdddddsssE dddssdssss sdddddsssss dddssdsssad 60 sssadssds Mighpay Dep.

<400> 14 ddssassds ssdasdadss yes cc dddddssed ssdssdads 60 ssisosad asdassisad EsEddssads saskhddddsd siddddssse asdesssss 120 sssddddssss ddssdassad ssadsseds Sdssdssss ssdsdaida ddssdsssssss 180 SSSSddasd sdaasadasas cDdadaass dddassss 220

<210> 15 <211> 2136 <212> DNA <213> Mig1pae dep.

<220>

<221> Coding sequence <222> (975) .. (1646) <400 15

dsddssdsda AEESDDSASD addsdsses dsedssassss dsdassesEa SESECESSESS 60 hdddd Esaaaaedu Eadsssssas shadadddas seadsseads sadsddddas 120 sda dddddddsd dssadd ssssdaaadd edddddedyyy sddddds 180 cddddss ssssdddaes Edddasdss dddddssdddaa ECEdasassd daeddaddy 240 zdgaddaddg Edsadaddda Eddad eddd SESDSSSS adasdsddss sdsdddssess 300 dgsadsaasa adESSssedd dssssadsss esdstdsdas Edddds adsssedsas 360 ssaadddssas adaddsed ssaaddssss ASEEEEEEAASE aaaadaddsd ssdssaddd 420 sasaeseskhd ddsa ACCESSORIES edddddaaad sssadsas desssaddad 480 addsssssad aaddasasdd assaddass SSSDSDSA dad surrender 540 dyddaaddaa sEsaadssas TACESESESS aassaessssd dsassssad sssedaadsa veg sadadsada ddddesEeada adasaddass AssadFed dadessessss ssEDaddssE 660 sadasdahst SEDADSESAD SEDADESEsd Eeesssses Edadadad adssa 720 SdassSeded dsa esddsdaa ddaasaddES edssaads ssSaasasad adadsaad 780 Bssassds adssassd dssdadssda Sessadssas SssaassEss Eddsssdsse 840 edadadassd dadeddaadd addaassass saaaddaeaaa SEAACESAEC EESSADEEEED 900 saadssddss saddaadadd dedddaaas dddssssasda addSeeEsda sdddadsses 960 Eddadssdaa adse aed daa sed dda seea dsa dad MeC C1i Ley C1u Ley A1a C1i fifteen sse ast dsa ee sss Rgo Thx A1a Lea Zeh 10 1010 sas ed sds ed ss ss add: dd garden ssa dss ed ΗΪ3 Suz Ley Agd Rgo Ahd Tgr C1p Zet A1a Tgr 15 20 Edd ssa ace Tgr Rgo Thx 25 : CEA DSE 'Bey A1a 1058

dss ssa dss sed sed hell Sds des asa daa des sss ssd das ssa aed 1106 ν · ι Bie thirty Aya Bie Bie Zeg Suz Wye TNg 35 Oyi A1a Zeg 40 Ley Azr Rgo Mes Sss sds hell sss des des sds das dde sss esa ssd dss ssd grandfather sss 1154 Veg 45 Agd Zeg Rgo Aya A1a fifty Agd Azr C1u Rgo Zeg 55 Mouth Wye Bie A1a it 60 sse asd das sas sed sse ddd dda sas ace grandfather eas sed sds hell yeah 1202 Echo TNG Azr Ayez Bie 65 Ego 31u Oyu H1z Th 70 A1a H1z Bie Suz Zeg 75 O1i hell ace sed food ses sed sss garden STS sse garden sss dsa sss ssd ssd 125C Agd TNG Bie Agd 80 Rgo Rgo Rgo Syp Zeg 85 Rgo Syp Rgo Aya RGS 90 Rgo Rgo Ltd dds sss grandfather sss garden sss sss sss dse grandfather = Ss sds ddd dsa sss 1298 EGO C1u Ego 95 Aya Bie Syp Zeg Rgo one hundred RGO Aya Aya Bie Agd 105 v1u A1a Agd grandfather grandfather eating dsa dda ace edd hell hell sds dsa edd ace asa das grandfather 1346 Aya Aya 110 Agd Aya C1u TNG Agd Zeg Zag Agd Aya Agd 120 TNG TNG Azr Aya sds dde eating sds sed sds sed ed sed grandfather ssd grandfather hell grandfather ses dds 1394 Agd 125 C1u Suz Agd Bie Agd 130 Zeg C1p Bie Ya1 Rgo 13 5 Ya1 Zeg Aya Bie C1u 140 ssa dde sas hell sss das dad sed asa sdS sss sds ess Vds hell dde 1442 Bie C1u Yayz Zeg Zeg 145 Avr 31and Bie Not Agd 150 Rae Agd Pb Suz Zeg 155 Zu esd eas sds food dsa sds ess garden sas das ses hell sed dss hell cSa 1490 Zeg Suz Agd Agd 16C Aya Agd Zeg Oyp H1z 155 Azr Bie Zeg Bie Aya 170 Zeg Bie sed dds dse ddd des cSa edd Sed sse sss I59 Sss edd sed ace hell 1535 Bie Oyu Aya 175 O1u A1a Bie Agd Zeg 190 Rgo Rgo □ 1y Zeg Agd 185 Rgo 11a Zeg garden sse Vds sds edd sss ace sds Sas dad dss dss sss ess aed das 1586 O1p Rgo 190 Suz Suz Agd Rgo TNG 195 Agd Tug O1i Aya Ya1 200 Zeg ? HB Nes Azr grandfather aas hell ace edd add ace grandfather das sas STS Sss dss ace dss sds 1634 Wye 205 Az η Zeg TNG Tgr Agd 210 Th Wye AZR ΗΪ3 Bie 215 Z A1a TNG Aya Suz 220 dds <31u sds souz csd ye dds oyu SdaddaSdaS ssasssssaa dssSeSdeas asSadassa 1686

sdedssdsss sasseddaas accessassassd ddesssessa assaddadss SsaasSsads 1746 addaedda ddsedsadad with addendsda dSdasadasd esdssddsaS 1806 dasadasada dsdaaadasd esddaassay Sdassaasad sessionadssd yeseddass 1866 osssssssas adasaddad aaessssadse aaadadaass sss ssddddad aaSssadaaaa 1926 sddsssssed Sssddddaa SdaaSSSSda adadaeaas aeasasasasas asacedad 1986 sdedeeds dassadss edcedaass adssssdedS ssassed aadssdaads 2046 cssessee assesaaaas eassesses sssedaaaaa aaasddssaa desddsskhss 2106

sSSSadSdad ddSSaaSSSSd SdaSsssddd <210> 16 <211> 224 <212> protein <213> Migaypay dep.

<400> 16

Month one Sy Bie Oyu Bie 5 Aya Oyy Rgo TNG Aya 10 Bie Zeg Ηίβ Suz Bie fifteen Agd RGO Agd Tgr Syp twenty Zeg Aya Tgr Tgr Rgo 25 TNG Bie Aya Wye Bie thirty Aya Bie Bie Zeg Suz 35 V "! Tag Oyy Aya Zeg 40 Bie Azr it MeC Zeg 45 Agd Zeg Rgo Aya Aya fifty Agd Azr Oyu Rgo Zeg 55 Rgo Wye Bie A1a Rgo 60 Rgo TNG Azr Nyz Bie 65 Rgo O1u Oyu H1z TNG 70 A1a ΗΪ5 Bie Suz Zeg 75 Oyy Agd TNG Bie Agd 80 Rgo Rgo Rgo 61p Zeg 85 Rgo C1G. Rgo Aya Ego 90 RGS Ego Rgo oh Rgo 95 Aya Bie Syp Zeg Rgo one hundred Rgo Aya Aya Bie Agd 105 Oyu Aya Agd A1a Aya no Agd Aya Oyu TNG Agd 115 Zeg Zeg Agd Aya Agd 120 Th TNG Azr Aya Agd 125 oh Suz Agd Bie Agd 130 Zeg Syp Bie Ya1 Rgo 13 5 Ya1 Zeg Aya Bie Oyu 140 Bie oh ΗΪ5 Zeg Zeg 145 Azr Sy Bie Not Agd 150 Pb Agd Pb Suz Zeg 155 O1u Zeg Suz Agd Agd 160 A1a Agd Zeg Oyp Nyz 165 Azr Bie Zeg Bie Aya 170 Zeg Bie Bie Oyu A1a 175 Oyu Aya Bie Agd Zeg 180 Rgo Rgo Oyu Zeg Agd 185 Rgo Not Zeg O1p RGO 190 Suz Suz Agd RGO TNG 195 Agd Tug O1i Aya Wye 2C0 Zeg Pb MeC Azr ν <1 205 AZP Zeg TNG Tgr Agd 210 Th Wye Azr She is 5 Bie 215 Zeg Aya TNG Aya suz 220 Oyu Suz Bie Oyu

<210> 17 <211> 18 <212> DNA <213> Synthetic sequence <220>

<223> Description of the synthetic sequence: PCR primer <400> 17 ccSddssads siasddd <210> <211> <212>

<213>

<220>

<223>

DNA

Synthetic sequence

Description of the synthetic sequence:

<400> 18 aaddadassd cSddsadsd <210>

<211>

<212>

<213>

<220>

<223>

dna

Synthetic sequence

Description of the synthetic sequence:

<400> 19 ASDDAASSSD DASSSDD <210> <211> <212>

<213>

<220>

<223>

DNA

Synthetic sequence

Description of the synthetic sequence:

<400> 20 cssassass assdds <210> <211> <212>

<213>

<220>

<223>

DNA

Synthetic sequence

Description of the synthetic sequence:

<400> 21 ddssassdss ssdasdad <210>

<211>

<212>

<213>

<220>

<223>

DNA

Synthetic sequence

Description of the synthetic sequence:

<400> ddsdddssss ddsessssad <210> <211> <212>

<213>

<220>

<223>

DNA

Synthetic sequence

Description of the synthetic sequence:

<400> 23 ssaadssssas sddddsdsss bssssssss <210>

<211>

<212>

<213>

<220>

<223>

DNA

Synthetic sequence

Description of the synthetic sequence:

<400> 24 saSassassas sddsadddds sssbssad <210> <211> <212>

<213>

<220>

<223>

DNA

Synthetic sequence

Description of the synthetic sequence:

<400> 25 dadssss sds ssddss ddas ssadssds ddas <210>

<211>

<212>

<213>

<220>

<223>

DNA

Synthetic sequence

Description of the synthetic sequence:

PCR primer

PCR primer

PCR primer

PCR primer

PCR primer

PCR primer

PCR primer

PCR primer

PCR primer <400> 26 cccssddssda ddddsdsdds SadDddddss sdsc <210> <211> <212> <213>

<220>

<223>

DNA

Synthetic sequence

Description of the synthetic sequence: hybridization probe <400> 27 psadddDes dddddddds Dssaadassd d <210> <211> <212>

<213>

DNA

Synthetic sequence <220>

<223>

Description of the synthetic sequence: PCR primer <210>

<211>

<212>

<213>

351

DNA

Noto zar1epz: 400> 29 asdds eddad dassdddd

СсдСдсдсдС Дсадсаддад сг1сдСдд = Unit ЕсдЭссддсдс: сСсаасСад ЕдссддСдсд

EdsasSdda sEdddasass deEssdasda assadsasdg

0 slssssssss:

DEESaddass ssdsadsgds. dsasDeSSs ssgsasda ce aEcEcEeads

180

EsEssasEad dadssddads assaadassd ssdssdddaS ssadass ds aEssssaassE

240

Edsadsadas: Sassada £ a sdaadsadEa EsEeEsasdd asdSaaa ss Easa Eddada

WSS Assdeaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

351 <210>

<211>

<212>

<213>

414

DNA

Noto zarDeps <400> 30 aEdddssaEc aaEsaEsaEsa

EsaEsaEsaE saEssessessa dsddssaeee sdasdasdas dasaaddsSd daddassddd aesssdEDsE sdsdsadssad dadsasdEDd ssdsssdeSed

123 sEdEssaas EadEddSddE dsdsdSasSs ddasDddDas AssdeEssda sdaassadsa

180 NEXT SESSION SESSION SESSION SESSION SESSION EECAECESSECA

240 dsEesEsEas Eaddadssdd adsasEaada ssdssdssdd dassEadass EdeaEsEsaa

300 ssEdeEdea dasseasead aeasdaadsa deaesseedes eddasdeaaa sesseasaedd

360 adaassdEad aEadaseEaec <210> <211> <212> <213>

Edsaassds

DNA

Synthetic: dSddeEd ~ s EddaEdaEa aEad sequence

414 <220>

<223>

Description of the synthetic sequence:

PCR primer <400> 31 aaddaaaaaa dsddssdssa eddaasEedd asEeddadd <210> <211> <212>

<213>

DNA

Synthetic sequence <220>

<223>

Description of the synthetic sequence:

primer <400>

Eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeepeet downseat sadssssadds

<213>

DNA

Synthetic sequence <220>

<223>

Description of the synthetic sequence:

primer <400> 33 dadsdadsss

Esadss

Claims (48)

FORMULA INVENTIONS 1. An isolated nucleic acid containing a nucleotide sequence selected from the group consisting of a) nucleotides 405-719 8EC ΙΌ ΝΟ: 1; b) a nucleotide sequence that specifically hybridizes with a sequence complementary to nucleotides 405-719 8EC ΙΌ ΝΟ: 1 under conditions of high stringency; where the nucleotide sequence encodes a neublastin polypeptide containing (ί) seven conserved cytheic residues at positions 8, 35, 39, 72, 73, 101 and 103 with numbering in accordance with 8ЕЕ ΙΌ ΝΟ: 2; (ίί) the following amino acid residues: C at position 8, b at position 10, V at position 17, b at position 20, Ο at position 21, b at position 22, Ο at position 23, E at position 28, at position 32, B at position 33, Ε at position 34, C at position 35, at position 37, C at position 39, C at position 72, C at position 73, B at position 74, P at position 75, Ε at position 83, at position 85, 8 at position 97, L at position 98, C at position 101 and C at position 103 when numbered in accordance with 8ETS ΝΟ: 2; (w) repeat ΕΟΕΟ, ΕBΕC motif, ORSSBR motif and 8ATA ^ C motif and (ίν) amino acid sequence with at least 90% homology with the LC! -LK ₁₀₅ 8EC sequence of Е ΝΟ: 2. 2. Nucleic acid containing a nucleic acid sequence that is complementary to the nucleic acid of claim 1. 3. A vector containing a nucleic acid according to claim 1. 4. The vector according to claim 3, where the specified vector is an expression vector. 5. The strain of cells transformed with a νίΐΐΌ vector according to claim 3 or 4. 6. The strain according to claim 5, where these cells are selected from the group consisting of mammalian cells, fungal cells, yeast cells, insect cells and bacteria cells. 7. The strain according to claim 6, where these cells are cells of the Chinese hamster ovary. 8. The strain according to claim 6, where these cells are cells derived from the central nervous system of a mammal. 9. A nucleic acid molecule encoding a neublastin polypeptide, wherein said nucleic acid molecule contains the sequence given in 8EC ΝΟ: 29 or 30. 10. A vector containing a nucleic acid according to claim 9. 11. The vector of claim 10, where the specified vector is an expression vector. 12. The strain of cells transformed with the νίΙΐΌ vector according to claim 10 or 11. 13. The strain indicated in paragraph 12, where these cells are bacterial cells. 14. An isolated neublastin polypeptide containing (a) seven conserved cytheic residues at positions 8, 35, 39, 72, 73, 101, and 103, when numbered in accordance with 8ЕЦ: 2; (b) the following amino acid residues: C at position 8, b at position 10, V at position 17, b at position 20, Ο at position 21, b at position 22, Ο at position 23, E at position 28, at position 32, B at position 33, Ε at position 34, C at position 35, at position 37, C at position 39, C at position 72, C at position 73, B at position 74, P at position 75, Ε at position 83, at position 85, 8 at position 97, L at position 98, C at position 101 and C at position 103 when numbered in accordance with 8ETS ΝΟ: 2; (c) repeat HCL, EKES motif, CRHCR motif and 8ATASS motif and (6) amino acid sequence with at least 90% homology with the sequence AK1-AK1 ₀₅ 8EC ΙΌ ΝΟ: 2. 15. The polypeptide of claim 14, wherein said polypeptide is glycosylated. 16. The polypeptide of claim 14, wherein said polypeptide is encoded by a nucleic acid according to claim 1 or 2. 17. The polypeptide according to any one of p-16, where the polypeptide has a C-terminal amino acid sequence represented by AK ₇₂ AK ₁₀₅ 8EO ΙΌ ΝΟ: 2. 18. The polypeptide according to any one of p-16, where the polypeptide has a C-terminal amino acid sequence represented by AK41AK ₁₀₅ 8EO ΙΌ ΝΟ: 2. 19. The polypeptide according to any one of p-16, where conservative amino acid substitutions are less than 10% of the total number of residues in the polypeptide. 20. The polypeptide according to claim 19, where conservative substitutions of amino acids constitute less than 2% of the polypeptide. 21. The polypeptide according to claim 19, where conservative amino acid substitutions are single amino acid substitutions in the sequence of the mature peptide, where both the replaced and the replacing amino acids are non-cyclic amino acids. 22. The peptide Neublastin, having any of the following sequences: ORSZVVAASAVSS (AK 30-43 ZEO Yu N0: 9); HONVOZEWUKES (AK 57-70 ZEO S N0: 9); SVKAZRNOZ (AK 74-85 3ΕΩ S N0: 9); ВВРРРСЗНРУЗОРС (АК 94-107 ЗЕО Ю N0: 9); ZTTUTOUKZATAS (AK 123-136 WE (2 S0 N0: 9); SKEKZAURURAVISSNVZOEUKEKGS (AK 43-70 ZEO S Y N0: 9); SKKAVZRNOZYAZYE ASIAVRRRBZKUUZORS (AK 74-107 ZEO S N0: 9) URVTUVAUUZEMOUYZTINTOUKZATAS (AK 108-136 ZEO 10 N0: 9); 0ΒΡΤΒΥΕΑν3ΕΜϋνΝ3Τ (AK 108-124 ZEO Yu N0: 9); and АККРРРСЗРУРУССРС (АК 93-107 ЗЕ5 Ю Н0: 9). 23. A composition comprising a polypeptide according to any one of paragraphs. 14-22 and a pharmaceutically acceptable carrier. 24. The method of administration of the polypeptide according to any one of paragraphs.14-16, providing for the stage of delivery of the specified polypeptide into a cell culture of v or w u1o in the body of a mammal. 25. The method of claim 24, wherein said administration comprises systemic administration. 26. The method of claim 24, wherein said mammal is affected by a condition selected from the group consisting of ischemic damage of brain neurons, traumatic brain damage, peripheral neuropathy, Alzheimer's disease, Tington's disease, Parkinson's disease, amyotrophic lateral sclerosis and memory impairment . 27. The method of claim 24, wherein said mammal is affected by a neuronal disorder of the peripheral nervous system, the medulla oblongata, or the spinal cord. 28. The use of a nucleic acid according to claim 1 or 2 for the manufacture of a medicament for the treatment of a neurodegenerative disease or disorder in an animal. 29. The use of the neublastin polypeptide according to any one of claims. 14-16 for the manufacture of a drug for the treatment of a neurodegenerative disease or disorder in an animal. 30. An antibody that specifically binds to any of the polypeptides of claim 14-16. 31. The antibody according to claim 30, wherein said antibody is a monoclonal antibody. 32. A method for determining the relationship of the presence of a neurodegenerative disease or disorder in an animal with an altered activity of the neublastin polypeptide neurotrophic factor, and this method includes the following stages: (a) contact of a biological sample obtained from said animal with an antibody according to claim 30 or 31, (b) determining the formation of an immune complex between said antibody and said protein; (c) comparing the content of the specified immune complex, which is formed in the specified sample, with the content of the specified immune complex, which is formed in the corresponding biological sample obtained from a patient not affected by the specified nervous condition, and (6) determining, based on the comparison, the connection of the specified disease or a disorder with the indicated altered level of activity of the neublastin polypeptide neurotrophic factor indicated. 33. A cell strain containing an exogenous regulatory sequence cloned in a functional relationship with the endogenous neublastin gene, the neublastin gene being able to encode the polypeptide of claim 14. 34. A method for producing a neublastin polypeptide according to any one of claims 14 to 22, wherein said method comprises culturing strain cells according to any one of claims 5 to 8, 12, 13 and 33 under conditions that allow the expression of said polypeptide. 35. The method according to clause 34, further comprising a stage of selection of the obtained polypeptide. 36. An isolated nucleic acid sequence containing the nucleotides 721-865 8ЕО ΙΌ ΝΟ: 1. 37. An antibody that specifically binds to any of the peptides described in item 22. 38. A kit that contains one or more containers with a substance selected from the group consisting of a) a neublastin polypeptide according to any one of claims 14-22; b) an antibody according to any one of claims 30, 31 and 37; c) one or more nucleotide probes capable of hybridizing with neublastin RNA; or d) at least one pair of nucleotide primers capable of initiating amplification of at least part of the neublastin gene. 39. A method for diagnosing or screening a subject or a person’s susceptibility to developing a disease or disorder that is characterized by the abnormal content of a neublastin polypeptide according to any one of claims 14-22, measuring the content of said neublastin polypeptide, neublastin polypeptide encoding RNA, or functional activity of the polypeptide neublastin in a sample obtained from the subject, with an increase or decrease in the content of the neublastin polypeptide, neublastin RNA or functional activity and the neublastin polypeptide in the sample relative to the content of the neublastin polypeptide, neublastin RNA or the functional activity of neublastin detected in a similar sample in the absence of a disease or disorder or in the absence of a predisposition to develop a disease or disorder, indicates the presence of a disease or disorder or susceptibility to develop a disease or disorder. 40. A method for screening a purified neublastin polypeptide according to any one of claims 14-22, or a derivative thereof, or a fragment thereof or a modulator of the activity of the above-mentioned peptides for activity in the treatment or prevention of a disease, this method comprising: (a) measuring changes in phenotype, genotype, behavior, survival or proliferation of cells from cell lines or a test animal, said cells or animal being produced in a disease state, or they exhibit signs associated with the disease, and said cells or animals interact with a neublastin polypeptide , its derivative, fragment or modulator, or it introduces the neublastin polypeptide, its derivative, fragment or modulator, and (b) comparing these changes with the phenotype, genotype, behavior, survives awn or proliferation of cells or animals which have not interacted with so neublastin polypeptide, a derivative thereof, or a fragment modulator, or not treated with the neublastin polypeptide, derivative, fragment, or modulator. 41. A method of treating peripheral neuropathies in a mammal, said method comprising administering a therapeutically effective amount of a neublastin polypeptide according to any one of claims 14-22, wherein said peripheral neuropathy is selected from the group consisting of neuropathies induced by injuries; chemotherapy-induced neuropathies; toxin-induced neuropathies; drug induced neuropathies; vitamin-induced neuropathies; idiopathic neuropathies and diabetic neuropathies. 42. The method of claim 25, wherein the neublastin is delivered directly to the central nervous system. 43. The method of claim 25, wherein the neublastin is delivered systemically by subcutaneous injection, intravenous injection or intravenous infusion. 44. A method for identifying, isolating, or determining new nucleic acid sequences, using a sequence of one or more nucleic acids of any one of claims 1, 2, and 36 in a computer program to identify, isolating, or identifying new nucleic acid sequences. 45. A fixed substrate or DNA chip that includes one or more nucleic acids according to any one of claims 1, 2 and 36. 46. A method for identifying, isolating, or determining new nucleic acid sequences involving the use of a fixed substrate or DNA chip according to claim 45. 47. A method for identifying, isolating, or determining new nucleic acid sequences, comprising using the sequence of one or more polypeptides of any of claims 14-22 in a computer program to identify, isolating, or identifying new nucleic acid sequences. 48. A method for identifying a compound that presumably induces a biological effect mediated by Neublastin, the method comprising the following steps: (a) obtaining a test cell, in which the expression of the product being determined is induced in said cell when interacting with the neublastin polypeptide according to any of claims 14-22; (b) the interaction of the cell with the test compound; and (c) identification of the product to be determined, moreover, said expression of the product being determined indicates the ability of the test compound to induce the specified neublastin-mediated biological effect.