WO2001094591A1 - A novel polypeptide-glyceraldehyde-3-phosphate dehydrogenase 10 and the polynucleotide encoding said polypeptide - Google Patents

Abstract

The invention discloses a new polypeptide-glyceraldehyde-3-phosphate dehydrogenase 10, the polynucleotide encoding said polypeptide, and a process for producing the polypeptide by recombinant DNA technology. It also discloses methods of applying the polypeptide for the treatment of various diseases, such as metabolism disorder, nervous disease, development disorder, tumor, inflammation, and immune disorders. This invention also discloses antagonist against said polypeptide and thereof therapy uses. In addition, it refers to the use of said polynucleotide encoding this new glyceraldehyde-3-phosphate dehydrogenase 10.

Description

 A new polypeptide monoglyceraldehyde-3-phosphate dehydrogenase 10 and a polynucleoside i encoding the polypeptide TECHNICAL FIELD

 The present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, glyceraldehyde-3-phosphate dehydrogenase 10, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a NAD-bound tetramer enzyme that is ubiquitous in glycolysis and gluconeogenesis (Harris LI Waters M The Enzymes (3rd edition) 13: 1-50 (1976)). In the middle of the protein molecule, a cysteine is involved in the formation of a covalent phosphorothioate intermediate. The sequence around the cysteine is completely conserved in GAPDH of eukaryotes and eukaryotes. The conserved characteristic sequence template is: [ASV]-S- C- [NT]-T- x (2)-[ LIM], where C (cysteine) is the active site residue (Zhao G., Pease AJ, Bharani N., Winkler ME, J. Bacteriol. 177: 2804-2812 (1995)) ₀

 GAPDH has many enzymatic and binding activities. Its main enzymatic activity exists as a uracil DNA glycosylase in glycolysis / gluconeogenesis (K. Meyer-Siegler et al., Proc. Natn. Acad. Sci. USA 88: 8460- 8464 (1991)). Other GAPDH functions include tRNA binding (R. Singh and M. Green, Science 259: 365-368 (1993)), and RNA-rich AU domains (E. Nagy and W. Rigby, J. Biol. Chem. 270 : 2755-2763 (1995)), combined with ATP (A. Nakai et al., Biochem. Biophys. Res. Coramun. 176: 59-64 (1991)) and calcium cyclin (F. Zeng et al., Int J, Biochem. 25: 1019-1027 (1993)). GAPDH forms a complex with the C-terminal domain of amyloid precursor protein (H. Schulze et al., J. Neurochera. 60: 1915-1922 (1993)). GAPDH also binds to eukaryotic glucose transporters (M. Lachaal et al., J. Biol. Chem. 265: 15449-15454 (1990)), and actin (C. Me jean et al., Biochem. J 264: 671-677 (1989)) and tubulin binding (M. Soraers et al., J. Eur. J. Biochem. 193: 437-444 (1990)). GAPDH binding to the expanded polyglutamine domain of a protein encoded by a CAG repeat domain gene can affect the enzyme or structural function of GAPDH and cause hereditary neurodegenerative diseases, leading to the death of fragile neurons and related to specific mental illness (A. Roses, J. NIH Res. 7: 51-57 (1995)).

The polypeptide of the present invention and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) family members have the same characteristic sequence template and have similar biological functions. Therefore, the polypeptide is considered to be glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ) Salary members of the family, the polypeptide and its inhibitors, agonists, antagonists are available for diagnosis and prevention as well as energy metabolism Related diseases and specific mental illnesses such as hereditary neurodegenerative diseases.

 Designation: Glyceraldehyde 3-phosphate dehydrogenase 10

 Since the glyceraldehyde 3-phosphate dehydrogenase 10 protein plays an important role in important functions of the body as described above, and it is believed that a large number of proteins are involved in these regulatory processes, there has been a need in the art to identify more glycerols involved in these processes. The aldehyde-3 -phosphate dehydrogenase 10 protein, in particular, identifies the amino acid sequence of this protein. Isolation of the neoglycerol-3-phosphate dehydrogenase 10 protein encoding gene also provides the basis for research to determine the role of this protein in health and disease states. This protein may form the basis for developing diagnostic and / or therapeutic drugs for diseases, so isolating its coding DNA is important. Disclosure of invention

 An object of the present invention is to provide an isolated novel polypeptide, monoglyceraldehyde-3-phosphate dehydrogenase 10, and fragments, analogs and derivatives thereof.

 Another object of the invention is to provide a polynucleotide encoding the polypeptide.

 Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a glyceraldehyde-3-phosphate dehydrogenase 10.

 It is another object of the present invention to provide a genetically engineered host cell containing a polynucleotide encoding a glyceraldehyde-3-phosphate dehydrogenase 10.

 Another object of the present invention is to provide a method for producing glyceraldehyde-3-phosphate dehydrogenase 10.

 Another object of the present invention is to provide an antibody against the polypeptide monoglyceraldehyde-3-phosphate dehydrogenase 10 of the present invention.

 Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors of the polypeptide monoglyceraldehyde-3-phosphate dehydrogenase 10 of the present invention.

 Another object of the present invention is to provide a method for diagnosing and treating a disease associated with an abnormality of glyceraldehyde 3-phosphate dehydrogenase 10.

 The present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof. Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.

 The invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:

 (a) a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID No. 2;

 (b) a polynucleotide complementary to polynucleotide (a);

(c) A polynucleotide having at least 70% identity to a polynucleotide sequence of (a) or (b). More preferably, the sequence of the polynucleotide is one selected from: (a) a sequence having positions 235-522 in SEQ ID NO: 1; and (b) having a sequence of 1-1745 in SEQ ID NO: 1 Sequence of bits.

 The invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.

 The invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.

 The invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of the glyceraldehyde 3-phosphate dehydrogenase 10 protein, which comprises utilizing the polypeptide of the invention. The invention also relates to compounds obtained by this method.

 The invention also relates to a method for detecting a disease or susceptibility to disease associated with abnormal expression of a glyceraldehyde-3-phosphate dehydrogenase 10 protein in vitro, which comprises detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample. Or detecting the amount or biological activity of a polypeptide of the invention in a biological sample.

 The invention also relates to a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.

 The present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of glyceraldehyde-3-phosphate dehydrogenase 10 .

 Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein.

 The following terms used in this specification and claims have the following meanings unless specifically stated: "Nucleic acid sequence" refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genome or a synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand. Similarly, the term "amino acid sequence" refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof. When the "amino acid sequence" in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide" or "protein" does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .

 A protein or polynucleotide "variant" refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. Variants can have "conservative" changes in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as the replacement of isoleucine with leucine. Variants can also have non-conservative changes, such as replacing glycine with tryptophan.

"Deletion" refers to the absence of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence. Missed.

 "Insertion" or "addition" means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature. "Replacement" refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.

 "Biological activity" refers to a protein that has the structure, regulation, or biochemical function of a natural molecule. Similarly, the term "immunologically active" refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response in appropriate animals or cells and to bind to specific antibodies.

 An "agonist" refers to a molecule that, when combined with glyceraldehyde 3-phosphate dehydrogenase 10, causes a change in the protein to regulate the activity of the protein. An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that binds glyceraldehyde-3-phosphate dehydrogenase 10.

 "Antagonist" or "inhibitor" refers to a biological activity or immunology that can block or regulate glyceraldehyde-3-phosphate dehydrogenase 10 when combined with glyceraldehyde 3-phosphate dehydrogenase 10 Active molecule. Antagonists and inhibitors can include proteins, nucleic acids, carbohydrates, or any other molecule that can bind glyceraldehyde 3-phosphate dehydrogenase 10.

 "Regulation" refers to a change in the function of glyceraldehyde-3-phosphate dehydrogenase 10, including an increase or decrease in protein activity, a change in binding characteristics, and any other biology of glyceraldehyde 3-phosphate dehydrogenase 10 Changes in nature, function, or immune properties.

 By "substantially pure" is meant substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify glyceraldehyde-3 -phosphate dehydrogenase 10 using standard protein purification techniques. Essentially pure glyceraldehyde-3-phosphate dehydrogenase 10 produces a single main band on a non-reducing polyacrylamide gel. The purity of the glyceraldehyde 3-phosphate dehydrogenase 10 peptide can be analyzed by amino acid sequence.

 "Complementary" or "complementary" refers to the natural binding of a nucleotide by base-pairing under conditions of acceptable salt concentration and temperature. For example, the sequence "C-T-G-A" can be combined with the complementary sequence "G-A-C-T". The complementarity between two single-stranded molecules may be partial or complete. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

"Homology" refers to the degree of complementarity and can be partially homologous or completely homologous. "Partial homology" refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. The inhibition of such hybridization can be detected by performing hybridization (Southern or Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction. "Percent identity" refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madison Wis.). The MEGALIGN program can compare two or more sequences according to different methods, such as the Clus ter method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). _{0 The} Clus ter method groups each group by checking the distance between all pairs. The sequences are arranged in clusters. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula:

 Number of matching residues between sequence A and sequence X 100

(The number of residues in sequence A-the number of spacer residues in sequence A-the number of spacer residues in sequence B) The percent identity between nucleic acid sequences can also be determined by the Clus ter method or by a method known in the art such as Jotun Hein ( Hein J., (1990) Methods in emzuraology 183: 625-645).

 "Similarity" refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences. Amino acids used for conservative substitutions, for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.

 "Antisense" refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence. "Antisense strand" refers to a nucleic acid strand that is complementary to the "sense strand".

 "Derivative" refers to a chemical modification of HFP or a nucleic acid encoding it. Such a chemical modification may be the replacement of a hydrogen atom with an alkyl group, an acyl group or an amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological characteristics of natural molecules.

"Antibody" refers to a complete antibody molecule and its fragments, such as Fa,? (^ ') ₂ and?, Which specifically bind to the epitope of glyceraldehyde-3-phosphate dehydrogenase 10.

 A "humanized antibody" refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.

The term "isolated" refers to the removal of a substance from its original environment (for example, its natural environment if it occurs naturally). For example, a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system. Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not in its natural environment Ingredients, they are still separate.

 As used herein, "isolated" refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment). For example, polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state. .

 As used herein, "isolated glyceraldehyde-3-phosphate dehydrogenase 10" means that glyceraldehyde-3-phosphate dehydrogenase 10 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify glyceraldehyde-3-phosphate dehydrogenase 10 using standard protein purification techniques. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the glyceraldehyde-3-phosphate dehydrogenase 10 polypeptide can be analyzed by amino acid sequence.

 The present invention provides a new polypeptide, glyceraldehyde-3-phosphate dehydrogenase 10, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2. The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide. The polypeptides of the present invention can be naturally purified products, or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude initial methionine residues.

 The invention also includes fragments, derivatives and analogs of glyceraldehyde-3-phosphate dehydrogenase 10. As used herein, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially retains the same biological function or activity of the glyceraldehyde-3 -phosphate dehydrogenase 10 of the present invention. A fragment, derivative, or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution The amino acid may or may not be encoded by a genetic codon; or (Π) such a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or (III) such One, in which the mature polypeptide is fused to another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV) such a polypeptide sequence in which the additional amino acid sequence is fused into the mature polypeptide ( Such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protease sequence) As explained herein, such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.

The present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2. The polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1. The polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 1745 bases in length and its open reading frame (235-522) encodes 87 amino acids. This peptide has There is a characteristic sequence of glyceraldehyde 3-phosphate dehydrogenase, and it can be deduced that the glyceraldehyde 3-phosphate dehydrogenase 10 has the structure and function represented by glyceraldehyde 3-phosphate dehydrogenase.

 The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. The DM can be a coding chain or a non-coding chain. The coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant. As used in the present invention, a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.

 The polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.

 The term "polynucleotide encoding a polypeptide" refers to a polynucleotide that includes the polypeptide and a polynucleotide that includes additional coding and / or non-coding sequences.

 The invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention. This polynucleotide variant can be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .

 The invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity between the two sequences). The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "strict conditions" means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficol l, 42 ° C, etc .; or (3) only between the two sequences Hybridization occurs only when the identity is at least 95%, and more preferably 97%. In addition, the hybridizable polynucleotide-encoded polypeptide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.

 The invention also relates to nucleic acid fragments that hybridize to the sequences described above. As used herein, a "nucleic acid fragment" contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nucleosides. Acid above. Nucleic acid fragments can also be used in nucleic acid amplification techniques such as PCR to identify and / or isolate polynucleotides encoding glyceraldehyde 3-phosphate dehydrogenase 10.

The polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity. The specific polynucleotide sequence encoding the glyceraldehyde-3-phosphate dehydrogenase 10 of the present invention can be obtained by various methods. For example, polynucleotides are isolated using hybridization techniques well known in the art. These technologies include but are not Limitations are: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleotide fragments with common structural characteristics.

 The DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.

 Of the methods mentioned above, genomic DM is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences. The standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature techniques for extracting mRNA, and kits are also commercially available (Qi agene). And the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Labora tory Manua, Co. Harbor Harbor Laboratory. New York, 1989). Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.

 The genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybrids; (2) the presence or absence of marker gene functions; (3) determination of the transcript of glyceraldehyde-3-phosphate dehydrogenase 10 Level; (4) detecting protein products of gene expression by immunological techniques or measuring biological activity. The above methods can be used singly or in combination.

 In the method (1), the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides. In addition, the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides. The probe used here is usually a DM sequence chemically synthesized based on the gene sequence information of the present invention. The genes or fragments of the present invention can of course be used as probes. DM probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).

 In the (4) method, immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product expressed by the glyceraldehyde-3-phosphate dehydrogenase 10 gene expression.

 A method using PCR technology to amplify DNA / RNA (Saik i, et al. Science 1985; 230: 1350-1 354) is preferably used to obtain the gene of the present invention. In particular, when it is difficult to obtain a full-length cDNA from a library, the RACE method (RACE-Rapid Amplification of cDNA Ends) can be preferably used. The primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods. The amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.

The polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be measured by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. To obtain the full-length cDNA sequence, The sequence needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length CDM sequence.

 The present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell that is genetically engineered using the vector of the present invention or directly using a glyceraldehyde-3-phosphate dehydrogenase 10 coding sequence, and that the present invention is produced by recombinant technology Methods of the polypeptide.

 In the present invention, a polynucleotide sequence encoding glyceraldehyde-3 -phosphate dehydrogenase 10 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention. The term "vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al. Gene, 1987, 56: 125) expressed in bacteria; pMSXND expression vectors expressed in mammalian cells (Lee and Na thans, JBio Chem. 263: 3521, 1988) and baculovirus-derived vectors expressed in insect cells. In short, as long as it can be replicated and stabilized in the host, any plasmid and vector can be used to construct a recombinant expression vector. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.

 Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding glyceraldehyde-3-phosphate dehydrogenase 10 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sarabroook, et al. Molecular Cloning, a Laboratory Manua, cold Harbor Harbor Laboratory. New York, 1989). The DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: E. coli lac or trp promoter; Lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoters , Retroviral LTRs and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenovirus enhancers.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture. Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli. Those of ordinary skill in the art will know how to select appropriate vector / transcription control elements (such as promoters, enhancers, etc.) and selectable marker genes.

 In the present invention, a polynucleotide encoding glyceraldehyde-3-phosphate dehydrogenase 10 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetic engineering containing the polynucleotide or the recombinant vector. Host cell. The term "host cell" refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E. coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as fly S2 or Sf 9; animal cells such as CH0, COS or Bowes melanoma cells.

Transformation of a host cell with a DNA sequence according to the present invention or a recombinant vector containing the DM sequence can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DM may be harvested after exponential growth phase, treated with CaC l ₂ method used in steps well known in the art. Alternatively, MgCl ₂ is used. If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.

 Using conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant glyceraldehyde 3-phosphate dehydrogenase 10 (Scence, 1984; 224: 1431). Generally there are the following steps:

 (1) using the polynucleotide (or variant) encoding human glyceraldehyde-3-phosphate dehydrogenase 10 of the present invention, or transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide;

 (2) culturing host cells in a suitable medium;

 (3) Isolate and purify protein from culture medium or cells.

 In step (2), depending on the host cell used, the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

In step (3), the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If desired, recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods. Brief description of the drawings

 The following drawings are used to illustrate specific embodiments of the invention, but not to limit the scope of the invention as defined by the claims.

 Fig. 1 is a comparison diagram of homology of the amino acid sequence of glyceraldehyde-3-phosphate dehydrogenase 10 of the present invention at a total of 46 amino acids at 63-109 and the characteristic domain of glyceraldehyde-3-phosphate dehydrogenase 10. The upper sequence is glyceraldehyde-3-phosphate dehydrogenase 10, and the lower sequence is the characteristic domain of glyceraldehyde 3-phosphate dehydrogenase. Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+".

 Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated glyceraldehyde-3-phosphate dehydrogenase 10. OkDa is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention

 The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are generally performed according to the conventional conditions such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Harbor Harbor Laboratory Press, 1989), or according to the manufacturing conditions Conditions recommended by the manufacturer. Example 1: Cloning of glyceraldehyde-3-phosphate dehydrogenase 10

 Total RM of human fetal brain was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. Poly (A) mRNA was isolated from total RNA using Quik mRNA Isolat ion Kit (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA. The Smart cDNA cloning kit (purchased from Clontech) was used to insert the cDNA fragments into the multicloning site of pBSK (+) vector (Clontech) to transform DH5 c. The bacteria formed a cDNA library. Dye terminate cycle reaction ion sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer) were used to determine the sequences at the 5 'and 3' ends of all clones. The determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 1214al2 was new DNA. The inserted cDNA fragment contained in this clone was determined in both directions by synthesizing a series of primers. The results show that the 1214al2 clone contains a full-length cDNA of 1745bp (as shown in Seq ID NO: 1), and has a 264bp open reading frame (0RF) from 235bp to 522bp, encoding a new protein (such as Seq ID NO : Shown in 2). We named this clone pBS-1214al2, and the encoded protein was named glyceraldehyde-3-phosphate dehydrogenase 10. Example 2: Domain analysis of cDNA clones

The sequence of the glyceraldehyde 3-phosphate dehydrogenase 10 of the present invention and the protein sequence encoded by the same are The prof i le scan program (BasiclocalAlgment search tool) [Altschul, SF et al. J. Mol. Biol. 1990; 215: 403-10] performs domain analysis in databases such as prosit. The glyceraldehyde-3-phosphate dehydrogenase 10 of the present invention is homologous with the domain glyceraldehyde-3-phosphate dehydrogenase at 63-109. The homology results are shown in FIG. 1, the homology rate is 20%, and the score is 9. 13; The threshold is 8. 81. Example 3: Cloning of a gene encoding glyceraldehyde-3-phosphate dehydrogenase 10 by RT-PCR

 CDNA was synthesized using fetal brain total RM as a template and ol igo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, the following primers were used for PCR amplification:

 Primerl: 5'- CATCCTGAGAACTGAAATTGATCGC- 3, (SEQ ID NO: 3)

 Primer 2: 5 '-ATAA A ATTTTTGAATTT ATGTTC A A- 3' (SEQ ID NO: 4)

 Primerl is a forward sequence starting at lbp of the 5th end of SEQ ID NO: 1;

 Pr imer2 is the 3'-end reverse sequence in SEQ ID NO: 1.

Conditions for the amplification reaction: 50 μl of Kol'L / Cl Kl '10 μl / L Tr is- CI, (pH 8.5.5), 1.5 mmol / L MgCl ₂ , 200 μ mol / L dNTP, l Opmol primer, 1U Taq DNA polymerase (Clontech). The reaction was performed on a PE9600 DM thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94. C 30sec; 55. C 30sec; 72 ° C 2min. During RT-PCR, set β-act in as a positive control and template blank as a negative control. The amplified product was purified using a QIAGEN kit and ligated to a pCR vector (Invitrogen) using a TA cloning kit. The DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1 to 1745bp shown in SEQ ID NO: 1. Example 4: Analysis of the expression of the glyceraldehyde-3-phosphate dehydrogenase 10 gene by Northern blot:

Total RM extraction in one step [Anal. Biochem 1987, 162, 156-159] ₀ This method involves acid guanidinium thiocyanate-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1) Centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water. With 20 g RNA, electrophoresed containing 20mM 3- EDTA-2. 2M on a 1.2% formaldehyde agarose gel (N- morpholino) propanesulfonic acid _(P H7. 0) -5raM sodium acetate -ImM . It was then transferred to a nitrocellulose membrane. A- ³² P dATP was used to prepare ³² P-labeled DNA probes by random primers. The DNA probe used was the PCR amplified glyceraldehyde-3-phosphate dehydrogenase 10 coding region sequence (235bp to 522bp) shown in FIG. 32P-labeled probes (approximately 2 x 10 ⁶ cpm / ml) were hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM H ₂ P0 ₄ (pH7. 4)-5 χ SSC- 5 χ Denhardt's solution and 200 μg / ml salmon sperm DNA. After hybridization, place the filter at 1 x SSC- 0.1% SDS in 55. C for 30 min. Then, Phosphor Imager was used for analysis and quantification. Example 5: In vitro expression, isolation and purification of recombinant glyceraldehyde 3-phosphate dehydrogenase 10

 According to SEQ ID NO: 1 and the coding region sequence shown in Figure 1, a pair of specific amplification primers was designed, the sequence is as follows:

Primer3: 5 -CCCCATATGATGCTCTGTCACCTTCAAAGGATGG-3 '(Seq ID No: 5) Priraer4: 5' -CCCAAGCTTCTTCAACATGCCGCTTCTGTTCTTC- 3 '(Seq ID No: 6) The 5' ends of these two primers contain Ndel and BamHI restriction sites, respectively, which The coding sequences of the 5 'and 3' ends of the gene of interest, respectively, and the Ndel and BamHI restriction sites correspond to the selectivity within the expression vector plasmid pET 28b (+) (Novagen, Cat. No. 69865. 3). Digestion site. The pBS-1214al2 plasmid containing the full-length target gene was used as a template for the PCR reaction. The PCR reaction conditions are: a total volume of 50 μ1, 10 pg of pBS-1214al2 plasmid, primers? 1 ^ [116: 1: -3 and? 1 ^ 11161: -4 points and another! | Is 1 (^ [1101, Advantage polymerase Mix (Clontech)) 1 μ 1. Cycle parameters: 94.C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 The digestion product and plasmid pET-28 (+) were double-digested with Ndel and BamHI, respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into the coliform bacteria DH5a by the calcium chloride method. After culturing overnight on LB plates containing kanamycin (final concentration 3 (^ _g / ral)), the positive clones were screened by colony PCR and sequenced. Positive clones (pET-1214al2) with the correct sequence were selected using calcium chloride. The recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) by using the method. In a LB liquid medium containing kanamycin (final concentration 30 μg / ml), the host strain BL21 (pET-1214al2) was 37 C. Cultivate to the logarithmic growth phase, add IPTG to a final concentration of 1 ol / L, and continue the cultivation for 5 hours. Centrifuge to collect the bacterial cells, ultrasonically break the bacteria, and centrifuge to collect the supernatant. of 3- & _§) bound affinity column 3. 8 (1 Quick Cartr idge ( Novagen Co.) by chromatography A purified target protein of glyceraldehyde-3 -phosphate dehydrogenase 10 was obtained. A single band was obtained at 10 OkDa by SDS-PAGE electrophoresis (Figure 2). The band was transferred to a PVDF membrane with Edams water The solution was analyzed by N-terminal amino acid sequence. As a result, the 15 amino acids at the N-terminus were completely identical to the 15 amino acid residues at the N-terminus shown in SEQ ID NO: 2. Example 6 Anti-glyceraldehyde 3-phosphate dehydrogenase 10 Production of antibodies

A peptide synthesizer (product of PE company) was used to synthesize the following peptides specific to glyceraldehyde-3 -phosphate dehydrogenase 10: Dragon 2-Met- Leu- Cys- His- Leu-Gln-Arg- Met-Va Bu Ser- Glu-Gln-Cys-His-Leu-C00H (SEQ ID NO: 7). The polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively. For methods, see: Avrameas, et al. Immunochemi s try, 1969; 6: 43. Rabbits were immunized with 4 mg of the hemocyanin peptide complex plus complete Freund's adjuvant, and 15 days later, hemocyanin peptide complex plus incomplete Freund's adjuvant Agent boosts immunity once. A titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum. Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose. The peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography. The immunoprecipitation method proved that the purified antibody could specifically bind to glyceraldehyde-3-phosphate dehydrogenase 10. Example 7: Use of a polynucleotide fragment of the present invention as a hybridization probe

 Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways. For example, the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected. Further, the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.

 The purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method. Acid sequence or a homologous polynucleotide sequence thereof. Filter hybridization methods include dot blotting, Southern imprinting, Northern blotting, and copying methods. They all use the same steps to immobilize the polynucleotide sample to be tested on the filter. These same steps are as follows: The sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer. The pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid. After the hybridization step, the unhybridized probes are removed by a series of membrane washing steps. This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature), so that the hybridization background is reduced and only strong specific signals are retained. The probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention The polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment. In this example, the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.

First, the selection of the probe

 The selection of oligonucleotide fragments for use as hybridization probes from the polynucleotide SEQ ID NO: 1 of the present invention should follow the following principles and several aspects to be considered:

1. The preferred range of probe size is 18-50 nucleotides;

2. GC content is 30% -70¾, if it exceeds, non-specific hybridization increases;

3. There should be no complementary regions inside the probe; 4. Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used;

5. Whether the preliminary selection probe is finally selected as a probe with practical application value should be further determined by experiments.

 After completing the above analysis, select and synthesize the following two probes:

 Probe 1 (probel), which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):

 5'-TGCAAGTACATTTGGAAAATCTGGTCTTGGCCCGTCCCCCA-3 '(SEQ ID NO: 8) Probe 2 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutation sequence (41Nt) of the gene fragment of SEQ ID NO: 1 or its complementary fragment:

 5 -TGCAAGTACATTTGGAAAATCTGGTCTTGGCCCGTCCCCCA- 3 '(SEQ ID NO: 9) For other commonly used reagents and their preparation methods related to the following specific experimental steps, please refer to the literature: DNA PROBES G. Keller; MM Manak; Stockton Press, 1989 (USA) and more commonly used molecular cloning laboratory manuals, such as the Guide to Molecular Cloning Experiments (Second Edition 1998) [US] Sambrook et al., Science Press.

 Sample Preparation:

 1.Extract DNA from fresh or frozen tissue

Steps: 1) Place fresh or freshly thawed normal liver tissue in a plate immersed in ice and filled with phosphate buffered saline (PBS). Cut the tissue into small pieces with scissors or a scalpel. Keep tissue moist during operation. 2) Centrifuge the tissue at 1,000 g for 10 minutes. 3) Use cold homogenization buffer (0.25mol / L sucrose; 25 legs ol / L Tris-HCl, pH 7.5; 25mraol / LnaCl; 25 legs ol / L MgCl ₂ ) suspension pellet (about 10ml / g X 4) Homogenize the tissue suspension at 4 ° C with an electric homogenizer at full speed until the tissue is completely broken. 5) Centrifuge at 1000g for 10 minutes. 6) Resuspend the cell pellet (1 to 5 ml per 0.1 g of the original tissue sample), and centrifuge at 1000 g for 10 minutes. 7) Resuspend the pellet in lysis buffer (1 ml per 0.1 g of the original tissue sample), and then follow the phenol extraction method below.

 2, DNA phenol extraction method

Steps: 1) Wash cells with 1-10 ml of cold PBS and centrifuge at 1000g for 10 minutes. 2) Resuspend the pelleted cells with cold cell lysate (lx 10 ⁸ cells / ml). Use a minimum of 100ul lysis buffer. 3) Add SDS to a final concentration of 1%. If SDS is directly added to the cell pellet before resuspending the cells, the cells may form large clumps that are difficult to break, and reduce the overall yield. This is particularly serious when extracting> 10 ⁷ cells. 4) Add proteinase K to a final concentration of 200ug / ml. 5) 50. C. Incubate for 1 hour or shake gently at 37 ° C overnight. 6) Extract with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and centrifuge in a small centrifuge tube for 10 minutes. The two should be clearly separated, otherwise centrifuge again. 7) Transfer the water phase to a new tube. 8) Extract with an equal volume of chloroform: isoamyl alcohol (24: 1) and centrifuge for 10 minutes. 9) Transfer the aqueous phase containing DM to a new tube. The DNA was then purified and ethanol precipitated.

3, DNA purification and ethanol precipitation

Steps: 1) Add 1/10 volume of 2mol / L sodium acetate and 2 volumes of cold 100% ethanol to the DNA solution and mix. Leave at -20 ° C for 1 hour or overnight. 2) Centrifuge for 10 minutes. 3) Carefully aspirate or pour out the ethanol. 4) Wash the pellet with 500ul of 70% cold ethanol and centrifuge for 5 minutes. 5) Carefully aspirate or pour out the ethanol. Wash the pellet with 500ul of cold ethanol and centrifuge for 5 minutes. 6) Carefully aspirate or pour out the ethanol, then invert on the absorbent paper to drain off the residual ethanol. Air dry for 10-15 minutes to allow the surface ethanol to evaporate. Be careful not to allow the pellet to dry completely, otherwise it will be more difficult to re-dissolve. 7) Resuspend the DNA pellet in a small volume of TE or water. Vortex at low speed or suck with a dropper while gradually increasing TE, mix until the DNA is fully dissolved, and add approximately 1 ul for each 1-5 χΐθ ⁶ cells.

 The following steps 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.

8) Add RNase A to the DNA solution to a final concentration of 100ug / ml, and incubate at 37 ° C for 30 minutes. 9) Add SDS and proteinase K to the final concentration of 0.5¾ and 100ug / ml, respectively. Incubate at 37 ° C for 30 minutes. 10) Extract the reaction solution with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and centrifuge for 10 minutes. 11) Carefully remove the aqueous phase and re-extract with an equal volume of chloroform: isoamyl alcohol (24: 1) and centrifuge for 10 minutes. 12) Carefully remove the aqueous phase, add 1/10 volume of 2mol / L sodium acetate and 2.5 volumes of cold ethanol, and mix well at -20 ° C for 1 hour. 13) Wash the pellet with 70% ethanol and 100% ethanol, air dry, and resuspend the nucleic acid. The process is the same as steps 3-6. 14) Measure A ₂₆ . And A ₂₈ . To detect the purity and yield of DNA. 15) Store at -20 ° C after dispensing. Preparation of sample film:

 1) Take 4x2 pieces of nitrocellulose membranes (NC membranes) of appropriate size, and mark the spotting position and sample number on it with a pencil. Two NC membranes are required for each probe, so that they can be used in the following experimental steps. The film was washed with high-strength conditions and strength conditions, respectively.

 2) Pipette and control 15 microliters each, spot on the sample film, and dry at room temperature.

 3) Place on filter paper impregnated with 0.1 mol / L NaOH, 1.5 mol / L NaCl for 5 minutes (twice), dry and place on filter paper impregnated with 0.5 mol / L Tris-HCl (pH 7.0), 3 mol / L NaCl Allow to dry for 5 minutes (twice).

 4) Clamped in clean filter paper, wrapped in aluminum foil, and dried under vacuum at 60-80 ° C for 2 hours.

 Labeling of probes

1) 3 μl Probe (0. IOD / Ιθ 1), add 2 μ IKinase buffer, 8-10 uCi ³² P-dATP + 2U Kinase, to make up to a final volume of 20 μ1.

 2) Incubate at 37 ° C for 2 hours.

 3) Add 1/5 volume of Bromophenol Blue Indicator (BPB).

 4) Pass Sephadex G-50 column.

5) Collect the first peak before ³² P-Probe washes out (can be monitored by Monitor).

 6) 5 drops / tube, collect 10-15 tubes.

 7) Monitor the amount of isotope with a liquid scintillator

8) were collected after the first peak were combined to ³² P- Probe (second peak to prepare the desired free γ- ³² P- dATP) ₀

 Pre-hybridization

 Put the sample film in a plastic bag, add 3-10 mg of pre-hybridization solution (lOxDenhardt's; 6xSSC, 0.1 lrag / ml CT DM (calf thymus DNA).), Seal the bag, and shake at 68 ° C for 2 hours in a water bath .

 Cross

 Cut a corner of the plastic bag, add the prepared probe, seal the bag, and shake at 42 ° C in water overnight. Wash film:

 High-intensity washing film:

 1) Take out the hybridized sample membrane.

 2) 2xSSC, 0.1 SDS, 40. C Wash for 15 minutes (twice).

 3) 0.1xSSC, 0.1% SDS, wash at 40 ° C for 15 minutes (twice).

 4) 0. IxSSC, 0.1% SDS, wash at 55 ° C for 30 minutes (twice), and dry at room temperature.

 Low-intensity washing film:

 1) Take out the hybridized sample membrane.

 2) 2xSSC, 0.1% SDS, wash at 37 ° C for 15 minutes (twice).

 3) Wash at 37 ° C for 15 minutes (twice) in IxSSC, 0. / oSDS.

 4) 0. IxSSC, 0.1% SDS, 40. Wash for 15 minutes (twice) and dry at room temperature.

 X-ray autoradiography:

 -70 ° C, X-ray autoradiography (press time depends on the radioactivity of the hybrid spot).

 Experimental results:

的 The hybridization experiments performed under low-intensity membrane washing conditions showed no significant difference in the radioactive intensity of the above two probe hybrid spots; while the hybridization experiments performed under high-intensity membrane washing conditions, the radioactive intensity of probe 1 was significantly stronger. To the radioactive intensity of the hybridization spot of another probe. Therefore, probe 1 can be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues. Industrial applicability

 The polypeptides of the present invention, as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.

 Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is ubiquitous in glycolysis and gluconeogenesis. GAPDH has many enzymatic and binding activities. Its main enzymatic activity exists as a uracil DNA glycosylase in glycolysis / gluconeogenesis. Other functions of GAPDH include binding to tRNA, binding to the RNA-rich AU domain, binding to ATP, and calcyclin. GAPDH forms a complex with the C-terminal domain of amyloid precursor protein. GAPDH also binds to eukaryotic glucose transporters, and to actin and tubulin. The binding of GAPDH to the expanded polyglutamine domain of a protein encoded by a gene with a CAG repeat domain can affect the enzyme or structural function of GAPDH and thus cause a genetic neurodegenerative disease, leading to the death of fragile neurons and to specific mental illness related.

 Glyceraldehyde-3-phosphate dehydrogenase-specific conserved sequences are required to form its active mot if.

 It can be seen that the abnormal expression of the specific glyceraldehyde 3-phosphate dehydrogenase family protein mot if will cause the function of the polypeptide containing the mot if of the present invention to be abnormal, thereby leading to the glycolysis regulated by the polypeptide of the present invention. As well as abnormalities in the gluconeogenesis pathway, the abnormalities of actin and tubulin that it regulates, the abnormalities of the cell cycle associated with calcyclin, especially the abnormal neuron function, and the death of fragile neurons. And related to specific mental illness.

It can be seen that the abnormal expression of glyceraldehyde 3-phosphate dehydrogenase 10 of the present invention will produce various diseases, especially disorders of energy and material metabolism, neurological diseases, developmental disorders, various tumors, inflammation, and immunity. Sexually transmitted diseases, including but not limited to:

 Disorders of energy and substance metabolism: isovaleric acidemia, propionic acidemia, methylmalonic aciduria, combined carboxylase deficiency, glutaric acid type I, phenylketonuria, mucolipid storage Disease, congenital lactose intolerance, hereditary fructose intolerance, galactosemia, defective fructose metabolism, glycogen storage disease

 Neurological diseases: Alzheimer's disease, Parkinson's disease, Chorea, Depression, Amnesia, Huntington's disease, Epilepsy, Migraine, Multiple sclerosis, Schizophrenia, Depression, Neurological decline

 Developmental disorders: congenital abortion, cleft palate, limb loss, limb differentiation disorder, atrial septal defect, neural tube defect, congenital hydrocephalus, mental retardation, skin, fat and muscular dysplasia, bone and joint development Adverse disease

Tumors of various tissues: gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumors, uterine fibroids, astrocytoma, ependymoma, glioblastoma, colon cancer, intrauterine Membrane cancer, colon cancer, thymic tumor, nasopharyngeal cancer, fibroid, fibrosarcoma Inflammation: chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, cerebrospinal multiple sclerosis, glomerulonephritis, myocarditis, cardiomyopathy, atherosclerosis, gastric ulcer, cervicitis, Various infectious inflammations

 Immune diseases: Systemic lupus erythematosus, rheumatoid arthritis, bronchial asthma, urticaria, specific dermatitis, post-infection myocarditis, scleroderma, myasthenia gravis, Guillain-Barre syndrome, common variable immunodeficiency disease , Primary B-lymphocyte immunodeficiency disease, Acquired immunodeficiency syndrome

 The abnormal expression of the glyceraldehyde 3-phosphate dehydrogenase 10 of the present invention will also produce certain hereditary, hematological diseases and the like.

 The polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially energy and material metabolism disorders, neurological diseases, development disorders, various Tumor, inflammation, immune disease, some hereditary, hematological diseases, etc.

 The invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) glyceraldehyde 3-phosphate dehydrogenase 10. Agonists increase the glyceraldehyde 3-phosphate dehydrogenase 10 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers. For example, mammalian cells or a membrane preparation expressing glyceraldehyde 3-phosphate dehydrogenase 10 can be cultured together with labeled glyceraldehyde 3-phosphate dehydrogenase 10 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.

 Antagonists of glyceraldehyde-3-phosphate dehydrogenase 10 include antibodies, compounds, receptor deletions, and the like that have been screened. An antagonist of glyceraldehyde-3-phosphate dehydrogenase 10 can bind to glyceraldehyde 3-phosphate dehydrogenase 10 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide Disabling the polypeptide from performing a biological function.

 When screening compounds as antagonists, glyceraldehyde-3-phosphate dehydrogenase 10 can be added to the bioanalytical assay. Effect to determine whether a compound is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same way as for screening compounds described above. Polypeptide molecules capable of binding to glyceraldehyde 3-phosphate dehydrogenase 10 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. During screening, the glyceraldehyde-3-phosphate dehydrogenase 10 molecule should generally be labeled.

The present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen. These antibodies can be polyclonal or monoclonal antibodies. The invention also provides antibodies against the glyceraldehyde-3-phosphate dehydrogenase 10 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries. Polyclonal antibodies can be produced by injecting glyceraldehyde-3-phosphate dehydrogenase 10 directly into immunized animals (such as rabbits, mice, rats, etc.). A variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant, etc. Techniques for preparing monoclonal antibodies to glyceraldehyde 3-phosphate dehydrogenase 10 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human beta -Cell hybridoma technology, EBV-hybridoma technology, etc. The chimeric human antibody constant region and the variable region of non-human origin may be used in combination Pat some production techniques (Morr i son et al, PNAS , 1985, 81: 6851) 0 Only some technical production of single chain antibodies (US Pat No. 4946778) can also be used to produce single chain antibodies against glyceraldehyde 3-phosphate dehydrogenase 10.

 Anti-glyceraldehyde-3-phosphate dehydrogenase 10 antibodies can be used in immunohistochemistry to detect glyceraldehyde-3-phosphate dehydrogenase 10 in biopsy specimens.

 Monoclonal antibodies that bind to glyceraldehyde-3-phosphate dehydrogenase 10 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.

 Antibodies can also be used to design immunotoxins that target a particular part of the body. For example, glyceraldehyde 3-phosphate dehydrogenase 10 high affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.). A common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill glyceraldehyde 3-phosphate dehydrogenase 10 positive cells.

 The antibodies of the present invention can be used for the treatment or prevention of diseases related to glyceraldehyde 3-phosphate dehydrogenase 10. Administration of an appropriate dose of antibody can stimulate or block the production or activity of glyceraldehyde 3-phosphate dehydrogenase 10.

 The invention also relates to a diagnostic test method for quantitatively and locally detecting the level of glyceraldehyde-3-phosphate dehydrogenase 10. These tests are well known in the art and include FISH assays and radioimmunoassays. The level of glyceraldehyde-3-phosphate dehydrogenase 10 detected in the test can be used to explain the importance of glyceraldehyde-3-phosphate dehydrogenase 10 in various diseases and to diagnose glyceraldehyde-3-phosphate dehydrogenase 10 Diseases where catalase 10 works.

 The polypeptide of the present invention can also be used for peptide mapping analysis. For example, the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.

The polynucleotide encoding glyceraldehyde 3-phosphate dehydrogenase 10 can also be used for a variety of therapeutic purposes. Gene therapy technology can be used to treat abnormal cell proliferation, development, or metabolism caused by the non-expression or abnormal / inactive expression of glyceraldehyde 3-phosphate dehydrogenase 10. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated glyceraldehyde 3-phosphate dehydrogenase 10 to inhibit endogenous glyceraldehyde 3-phosphate dehydrogenase 1 0 activity. For example, a variant glyceraldehyde 3-phosphate dehydrogenase 10 may be a shortened glyceraldehyde 3-phosphate dehydrogenase 10 lacking a signaling domain, although it can bind to a downstream substrate, However, it lacks signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of glyceraldehyde-3-phosphate dehydrogenase 10. Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, and the like can be used to transfer a polynucleotide encoding glyceraldehyde-3-phosphate dehydrogenase 10 into a cell. A method for constructing a recombinant viral vector carrying a polynucleotide encoding a glyceraldehyde-3-phosphate dehydrogenase 10 can be found in the existing literature (Sambrook, et al.). In addition, a recombinant polynucleotide encoding glyceraldehyde-3-phosphate dehydrogenase 10 can be packaged into liposomes and transferred into cells.

 Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.

 Oligonucleotides (including antisense RNA and DNA) and ribozymes that inhibit glyceraldehyde 3-phosphate dehydrogenase 10 raRNA are also within the scope of the present invention. A ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific MA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation. Antisense RM, DNA and ribozymes can be obtained by any RNA or DNA synthesis technology. For example, solid-phase phosphoramidite chemical synthesis technology has been widely used. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of the DM sequence encoding the RM. This DM sequence has been integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.

 The polynucleotide encoding glyceraldehyde-3-phosphate dehydrogenase 10 can be used for the diagnosis of diseases related to glyceraldehyde-3-phosphate dehydrogenase 10. A polynucleotide encoding glyceraldehyde 3-phosphate dehydrogenase 1 0 can be used to detect the expression of glyceraldehyde 3-phosphate dehydrogenase 1 0 or glyceraldehyde 3-phosphate dehydrogenase 1 0 in a disease state Abnormal expression. For example, the DM sequence encoding glyceraldehyde-3-phosphate dehydrogenase 10 can be used to hybridize biopsy specimens to determine the expression of glyceraldehyde-3-phosphate dehydrogenase 10. Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and the relevant kits are commercially available. Some or all of the polynucleotides of the present invention can be used as probes to be fixed on a micro array (Mi croar ray) or a DNA chip (also known as a "gene chip") for analyzing differential expression analysis of genes and genetic diagnosis in tissues . Glyceraldehyde-3 phosphate dehydrogenase 10 specific primers can be used for RNA-polymerase chain reaction (RT-PCR) in vitro amplification to detect the transcription products of glyceraldehyde 3-phosphate dehydrogenase 10.

Detecting glyceraldehyde 3-phosphate dehydrogenase 10 gene mutations can also be used to diagnose glyceraldehyde 3-phosphate dehydrogenase Enzyme 10 related diseases. The forms of the glyceraldehyde-3-phosphate dehydrogenase 10 mutation include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type glyceraldehyde 3-phosphate dehydrogenase 10 DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DM sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.

 The sequences of the invention are also valuable for chromosome identification. The sequence specifically targets a specific position on a human chromosome and can hybridize to it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.

 In short, PCR primers (preferably 15-35bp) are prepared according to cDM, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.

 PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes. Using the oligonucleotide primers of the present invention, in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization. Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.

 Fluorescent in situ hybridization (FISH) of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step. For a review of this technique, see Verma et al., Human Chromosomes: a Manua l of Basic Techniques, Pergamon Pres s, New York (1988).

 Once the sequence is located at the exact chromosomal location, the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendelian Inheritance in Man (available online with Johns Hopkins University Wetch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.

Next, the differences in cDNA or genomic sequences between the affected and unaffected individuals need to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable using cDNA sequence-based PCR. Based on the resolution capabilities of current physical mapping and gene mapping technologies, cDNAs that are accurately mapped to disease-related chromosomal regions can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping capability and every 20kb corresponds to a gene).

 The polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier. These carriers can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof. The composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.

 The invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention. Along with these containers, there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell. In addition, the polypeptides of the invention can be used in combination with other therapeutic compounds.

 The pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration. Glyceraldehyde-3 phosphate dehydrogenase 10 is administered in an amount effective to treat and / or prevent a specific indication. The amount and range of glyceraldehyde-3-phosphate dehydrogenase 10 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

Claims

Claim

1. An isolated polypeptide-glyceraldehyde-3-phosphate dehydrogenase 10, characterized in that it comprises: a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, or an active fragment, analog, or derivative thereof Thing.

 2. The polypeptide according to claim 1, characterized in that the amino acid sequence of the polypeptide, analog or derivative has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 2.

 3. The polypeptide according to claim 2, characterized in that it comprises a polypeptide having the amino acid sequence shown in SEQ ID NO: 2.

 4. An isolated polynucleotide, characterized in that said polynucleotide comprises one selected from the group consisting of:

 (a) a polynucleotide encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or a fragment, analog, or derivative thereof;

 (b) a polynucleotide complementary to polynucleotide (a); or

 (c) A polynucleotide that is at least 70% identical to (a) or (b).

 5. The polynucleotide according to claim 4, wherein the polynucleotide comprises a polynucleotide encoding an amino acid sequence represented by SEQ ID NO: 2.

 6. The polynucleotide according to claim 4, characterized in that the sequence of the polynucleotide comprises the sequence of positions 235-522 in SEQ ID NO: 1 or the sequence of positions 1-1745 in SEQ ID NO: 1. .

 7. A recombination vector containing an exogenous polynucleotide, characterized in that it is a recombination constructed by the polynucleotide according to any one of claims 4-6 and a plasmid, virus or a carrier expression vector Carrier.

 8. A genetically engineered host cell containing an exogenous polynucleotide, characterized in that it is selected from one of the following host cells:

 (a) a host cell transformed or transduced with the recombinant vector of claim 7; or

 (b) a host cell transformed or transduced with the polynucleotide according to any one of claims 4-6.

9. A method for preparing a polypeptide having glyceraldehyde-3-phosphate dehydrogenase 10 activity, characterized in that the method includes: ''

 (a) culturing the engineered host cell of claim 8 under conditions in which glyceraldehyde-3-phosphate dehydrogenase 10 is expressed;

 (b) A polypeptide having glyceraldehyde-3 -phosphate dehydrogenase 10 activity is isolated from the culture.

 10. An antibody capable of binding to a polypeptide, characterized in that the antibody is an antibody capable of specifically binding to glyceraldehyde-3-phosphate dehydrogenase 10.

11. A class of compounds that mimic or regulate the activity or expression of a polypeptide, characterized in that they are compounds that mimic, promote, antagonize or inhibit the activity of glyceraldehyde 3-phosphate dehydrogenase 10.

12. The compound according to claim 11, characterized in that it is an antisense sequence of the polynucleotide sequence shown in SEQ ID NO: 1 or a fragment thereof.

 13. Use of a compound according to claim 11, characterized in that the compound is used for a method for regulating the activity of glyceraldehyde-3 -phosphate dehydrogenase 10 in vivo and in vitro.

 14. A method for detecting a disease or susceptibility to a disease associated with a polypeptide according to any one of claims 1-3, characterized in that it comprises detecting the expression level of the polypeptide, or detecting the activity of the polypeptide Or detecting a nucleotide variation in a polynucleotide that causes abnormal expression or activity of the polypeptide.

 15. The use of the polypeptide according to any one of claims 1-3, characterized in that it is used for screening mimetics, agonists, antagonists or inhibitors of glyceraldehyde-3-phosphate dehydrogenase 10 ; Or for identification of peptide fingerprints.

 16. The use of a nucleic acid molecule according to any one of claims 4-6, characterized in that it is used as a primer for a nucleic acid amplification reaction, or as a probe for a hybridization reaction, or used for manufacturing a gene chip. Or microarray.

 17. Use of a polypeptide, polynucleotide or compound according to any one of claims 1-6 and 11, characterized in that said polypeptide, polynucleotide or mimetic, agonist, antagonist is used Or the inhibitor is composed of a safe and effective dose with a pharmaceutically acceptable carrier as a pharmaceutical composition for diagnosing or treating a disease associated with an abnormality of glyceraldehyde 3-phosphate dehydrogenase 10.

 18. The use of a polypeptide, polynucleotide or compound according to any one of claims 1-6 and 11, characterized in that the polypeptide, polynucleotide or compound is used for preparing for treating malignant tumors, blood, etc. Diseases, HIV infection and immune diseases and drugs of various inflammations.