WO2001040297A1 - A novel polypeptide - human atp/gtp binding domain-containing transition nuclear protein 10 and a polynucleotide encoding the same - Google Patents

Abstract

The present invention discloses a novel polypeptide - human ATP/GTP binding domain-containing transition nuclear protein 10 and a polynucleotide encoding the same, as well as a method of producing the polypeptide by DNA recombinant technology. The present invention also discloses methods of using the polypeptide in treatment of various disease, such as malignant tumor, blood disease, HIV infection, immunological disease and various inflammation. The present invention also discloses an antagonist against the polypeptide and the therapeutic use of the same. The present invention also discloses the use of such polynucleotide encoding human ATP/GTP binding domain-containing transition nuclear protein 10.

Description

 A new multi-ft-human shuttle-converting protein 10 containing an ATP / GTP binding domain and a polynucleotide encoding the polypeptide TECHNICAL FIELD

 The present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, a human nuclear transfer protein 10 containing an ATP / GTP binding domain, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique

 Sperm formation is a process in which a gamete is gradually differentiated and finally forms a mature sperm cell. In mammals, the first stage of sperm formation is the beginning of sperm cell development, and this stage is related to changes in chromatin structure; the second stage is to form a complete, transcriptionally inactive non-nucleus through fusion and aggregation of chromatin Body structure. This fusion process involves the transformation of two proteins. The first transformation is a histidine transformation and replacement process mediated by many sperm cell-specific proteins, and these proteins are replaced by one or two prolines in the second transformation process. These sperm cell-specific proteins play an important regulatory role in the second stage of mature sperm formation. Abnormal expression of these proteins will cause individuals to fail to form normal, mature sperm, which will cause various related reproductive system diseases [Frederic CHIRAT , Arlette MARTINAGE et al., Eur. J. Biochem. 1991, 198: 13-20].

 People have long cloned TP1, TP2, TP3, and TP4 from rat and mouse. Among them, the study of TP1 and TP2 is more detailed. Subsequently, human and bovine TP 1 proteins similar to the mouse TP1 protein were cloned from humans and cattle. The bovine TP1 protein contains a cysteine residue, while the human TP1 protein is similar to the mouse TP1 and does not contain a cysteine residue. These proteins all play important regulatory roles in the spermatogenesis of these organisms.

 Nuclear transfer protein 1 is one of the sperm cell-specific proteins. The conversion protein consists of 54 amino acid residues, and its sequence is highly conserved in mammals. We found a 7 amino acid long signal fragment at the 28th to 34th amino acid positions. The amino acid sequence of this fragment is as follows: S-K-R-K-Y-R-K. This sequence fragment contains a tyrosine residue and four serine residues. These four sites are the sites of protein phosphorylation. Among them, the tyrosine site is responsible for chromatin during protein-MA interaction. Instability is required; and the phosphorylation of the serine site is also necessary for protein-DNA interactions, which may regulate the correct binding of the protein to specific sites in the DNA. The abnormal expression of this domain will directly lead to the abnormal function of the protein during spermatogenesis, which will cause various phases of reproductive system diseases.

ATP or GTP binding motifs are ubiquitous domains in various ATP / GTP binding proteins. Some are connected to ATP Both GTP and GTP proteins more or less contain this conserved mot if sequence, which has a conserved characteristic sequence fragment: [AG]-X (4)-G- K- [ST] (where X is arbitrary Amino acid residues). The most conserved of these mot if is the gl yc i ne enriched region, which forms a flexible loop between the (3-line and the alpha helix. This curved loop is usually associated with a phosphate group on the nucleotide Group reaction, the mot if sequence is usually called "A" consensus sequence or P loop. This motif is usually responsible for binding with ATP and GTP in the organism, providing the energy required for the protein to perform biological functions and stabilizing the protein in the organism. The structure of the body assists the protein to complete its normal physiological function. Abnormal expression will directly lead to the abnormal expression and function of some proteins, which will cause developmental disorders of various tissues, tumors and cancers.

 The new human nuclear transfer protein 10 containing an ATP / GTP binding domain is similar to other nuclear transfer proteins. It also contains a conserved signal fragment consisting of 7 amino acid residues and contains an ATP / GTP binding domain. Therefore, this protein is a new type of human nuclear conversion protein containing ATP / GTP binding domain, and is similar to other nuclear conversion proteins, has similar biological functions, and is closely related to the occurrence of some reproductive system diseases in vivo.

 Since the human ATP / GTP binding domain-containing nuclear transfer protein 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, more needs to be identified in the art Processes the human ATP / GTP binding domain-containing nuclear transfer protein 10 protein, and in particular the amino acid sequence of this protein is identified. Isolation of the ATP / GTP binding domain-containing nuclear conversion protein 10 protein encoding genes in newcomers also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so isolating its coding DNA is important. Disclosure of invention

It is an object of the present invention to provide isolated novel polypeptides-human nuclear conversion proteins 10 containing ATP / GTP binding domains, 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 human ATP / GTP binding domain-containing nuclear conversion protein 10.

 Another object of the present invention is to provide a genetically engineered host cell containing 10% nucleotides encoding a human ATP / GTP binding domain-containing nuclear conversion protein.

 Another object of the present invention is to provide a method for producing a human ATP / GTP binding domain-containing nuclear conversion protein 10. Another object of the present invention is to provide an antibody against a human-to-human ATP / GTP binding domain-containing nuclear conversion protein 10 of the polypeptide of the present invention.

 Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors of the nuclear transfer protein 10 containing ATP / GTP binding domains of the polypeptide of the present invention.

Another object of the present invention is to provide diagnosis and treatment of human ATP / GTP binding domain-containing nuclear converter protein 10 abnormal phase. Related diseases.

 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 the group consisting of: (a) a sequence having positions 206-469 in SEQ ID NO: 1; and (b) a sequence having 1-577 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 present invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit human ATP / GTP binding domain-containing nuclear transduction protein 10 protein activity, which comprises utilizing the polypeptide of the present invention. The invention also relates to compounds obtained by this method.

 The invention also relates to a method for in vitro detection of a disease or disease susceptibility related to abnormal expression of a nuclear conversion protein 10 protein containing human ATP / GTP binding domain, which comprises detecting the polypeptide or a polynucleotide encoding the same in a biological sample. A mutation in a sequence, or the amount or biological activity of a polypeptide of the invention in a biological sample is detected.

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

 The present invention also relates to the preparation of polypeptides and / or polynucleotides of the present invention for the treatment of some reproductive system diseases, developmental disorders of related tissues, tumors and cancers or other nuclear conversion proteins due to humans containing an ATP / GTP binding domain. 10 Use of a medicine for diseases caused by abnormal expression.

 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 otherwise:

"Nucleic acid sequence" refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and can also refer to genomic or synthetic Ww \ or RNA, which can be single-stranded or double-stranded, representing the sense strand 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 When "column" relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide" or "protein" is not meant to limit the amino acid sequence to the complete natural amino acid associated with 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, which may have "conservative" changes, wherein the substituted amino acid has a structural or chemical property similar to the original amino acid, Such as replacing leucine with leucine. Variants can also have non-conservative changes, such as replacing glycine with tryptophan.

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

 "Insertion" or "addition" refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule. "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 "immunological activity" refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind to specific antibodies in a suitable animal or cell.

 An "agonist" refers to a molecule that, when combined with a human ATP / GTP binding domain-containing nuclear conversion protein 10, can cause the protein to change, thereby regulating the activity of the protein. An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that binds to a human ATP / GTP binding domain-containing nuclear conversion protein 10.

 An "antagonist" or "inhibitor" refers to a nuclear conversion protein that can block or regulate human ATP / GTP binding domain-containing 1 when it binds to a human ATP / GTP binding domain-containing nuclear conversion protein. A biologically or immunologically active molecule. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that binds to a human ATP / GTP binding domain-containing nuclear conversion protein 10.

 "Regulation" refers to a change in the function of a human ATP / GTP binding domain-containing nuclear conversion protein 10, including an increase or decrease in protein activity, a change in binding characteristics, and a human ATP / GTP binding domain-containing nuclear conversion protein. Any other biological, functional, or immunological changes.

 "Substantially pure" means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify human ATP / GTP binding domain-containing nuclear conversion proteins 10 using standard protein purification techniques. Substantially pure human ATP / GTP binding domain-containing nuclear transfer protein 10 produces a single main band on a non-reducing polyacrylamide gel. The purity of the human ATP / GTP binding domain-containing nucleoprotein 10 peptide can be analyzed by amino acid sequence.

"Complementary" or "complementarity" refers to polynucleotides that are base-paired by allowable salt concentration and temperature conditions Ran combined. For example, the sequence "C-TGA" can be combined with the complementary sequence "GA-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. This inhibition of hybridization can be detected by performing hybridization (Southern blotting or Nor thern 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 two sequences bind to each other as a specific or selective interaction.

 "Percent identity" refers to the percentage of sequences that are the same 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., Mad Son Wis.). The MEGALIGN program can compare two or more sequences (Higgins, D. G. and P. M. Sharp (1988) Gene 73: 237-244) according to different methods, such as the Clus ter method. The C l uster method arranges each group of sequences into clusters by checking the distance between all pairs. 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 B

 1 00 Number of residues in sequence A-number of interval residues in sequence A-number of interval residues in sequence B

 The percent identity between nucleic acid sequences can also be determined by the Clus ter method or by methods known in the art such as Jotun He in (He in J., (1990) Methods i n emzumo logy 183: 625-645).

 "Similarity" refers to the degree to which the amino acid residues at the corresponding positions are replaced by the same or conservative substitutions in the alignment between 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 a "sense strand."

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

"Antibody" refers to a complete antibody molecule and its fragments, such as Fa, 卩 (^ ') ₂ and 1 ^, which can specifically bind human Antigenic determinants of ATP / GTP binding domain of nuclear conversion protein 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 vector, or such a polynucleotide or polypeptide may be part of a composition. Since the carrier or composition is not a component of its natural environment, they are still isolated.

 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 human ATP / GTP binding domain-containing nuclear conversion protein 1 0" refers to human ATP / GTP binding domain-containing nuclear conversion protein 1 0 that is substantially free of other proteins and lipids naturally associated with it. Class, sugar or other substance. Those skilled in the art can purify human ATP / GTP binding domain-containing nuclear conversion proteins 10 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the human ATP / GTP binding domain-containing nucleoprotein 10 peptide can be analyzed by amino acid sequence.

 The present invention provides a novel polypeptide-to-human nuclear conversion protein 10 containing an ATP / GTP binding domain, which basically consists of the amino acid sequence shown in SEQ ID D N0: 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 starting methionine residues.

The present invention also includes fragments, derivatives, and analogs of human ATP / GTP binding domain-containing nuclear conversion protein 10. As used herein, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially maintains the same biological function or activity of the human ATP / GTP binding domain-containing nuclear conversion protein 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 replaced 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 the genetic code; or (II) such a type in which a group on one or more amino acid residues is substituted by other groups to include a substituent; or (III) such One in which the mature peptide is in combination with another compound (such as A peptide half-life compound, such as polyethylene glycol), or (IV) a polypeptide sequence (such as a leader sequence or a secreted sequence or a sequence used to purify the polypeptide) in which an additional amino acid sequence is fused into a mature polypeptide (Or proteomic sequences) As set forth 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 with a total length of 579 bases, and its open reading frame (206-469) encodes 87 amino acids. This polypeptide has a characteristic sequence of a nuclear conversion protein and an ATP / GTP binding domain. It can be deduced that the human nuclear conversion protein 10 containing an ATP / GTP binding domain has the structure and function represented by the nuclear conversion protein.

 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. MA can be coded or non-coded. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant. As used herein, 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 comprising the polypeptide and a polynucleotide comprising 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. Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. 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 invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the 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) adding a denaturant during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1 »/» F i co ll, 42 "C, etc .; or ( 3) Only the identity between the two sequences is at least 95%, Hybridization occurs only when it is above 97%. In addition, the polypeptide encoded by the hybridizable polynucleotide 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 in the present invention, 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 cores. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding human ATP / GTP binding domain-containing nucleoprotein 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 of the present invention encoding human ATP / GTP binding domain-containing nuclear conversion protein 10 can be obtained by various methods. For example, polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.

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

 Of the methods mentioned above, genomic DNA isolation 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 mRNA extraction, and kits are also commercially available (Qiagene). Construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989). Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When combined with polymerase reaction technology, even very few 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): (DDNA-DA or DNA-RNA hybridization; (2) the presence or absence of a marker gene function; (3) determination of human ATP / GTP binding domain-containing nuclear transfer protein 10 transcription The current level; (4) Detecting the protein product of gene expression by immunological techniques or measuring biological activity. The above method can be used alone 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 2,000 nucleotides, preferably within 1000 nucleotides. The probes used here are usually chemically synthesized based on the gene sequence information of the present invention. sequence. The genes or fragments of the present invention can of course be used as probes. DNA probes are labeled with radiation Sexual isotopes, luciferin or enzymes (such as alkaline phosphatase).

 In the (4) method, the protein product of the human ATP / GTP binding domain-containing nuclear conversion protein 10 gene expression can be detected using immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). Wait.

 A method of amplifying DNA / RNA by PCR (Saiki, et al. Science 1985; 230: 1350-1354) 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) may be preferably used. The primers used for PCR may 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 separated 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 determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequencing can also be performed using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.

 The present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a human ATP / GTP binding domain-containing nuclear conversion protein 10 coding sequence, and produced by recombinant technology A method of a polypeptide according to the invention.

 In the present invention, a polynucleotide sequence encoding a human ATP / GTP binding domain-containing nuclear conversion protein 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, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors 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; MSXND expression vectors expressed in mammalian cells ( Lee and Nathans, J Bio Chem. 263: 3521, 1988) and baculovirus-derived vectors expressed in insect cells. In short, as long as it can be replicated and stabilized in a 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 well known to those skilled in the art can be used to construct expression vectors containing MA sequences encoding human ATP / GTP binding domain-containing nuclear transfer protein 10 and suitable transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989). The DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. These start Representative examples of promoters are: l ac or trp promoter of E. coli; PL promoter of lambda phage; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, anti LTRs that transcribe viruses 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 and a transcription terminator for translation initiation. 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. Examples include 100 to 270 base pair SV40 enhancers on the late side of the origin of replication, polyoma enhancers and adenovirus enhancers on the late side of the origin of replication.

 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 regulatory elements (such as promoters, enhancers, etc.) and selectable marker genes.

 In the present invention, a polynucleotide encoding a human ATP / GTP binding domain-containing nuclear conversion protein 10 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a polynucleotide containing the polynucleotide or the recombinant vector. Genetically engineered host cells. 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: Escherichia 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 described in the present invention or a recombinant vector containing the DNA 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 DNA can be harvested after the exponential growth phase and treated with the CaCl ₂ method. The steps used are 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.

 The polynucleotide sequence of the present invention can be used to express or produce recombinant human ATP / GTP binding domain-containing nuclear conversion protein 10 by conventional recombinant DNA technology (Scence, 1984; 224: 1431). Generally there are the following steps:

(1) A polynucleotide (or variant) encoding a human-human ATP / GTP binding domain-containing nuclear conversion protein 10 of the present invention, or a recombinant expression vector containing the polynucleotide for transformation or transduction Host cell: (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 media. 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 necessary, the physical, chemical, and other properties can be used to separate and purify the recombinant protein by various separation methods. 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 the amino acid sequence homology of the nuclear transfer protein 10 containing the ATP / GTP binding domain of the present inventor with 47 amino acids in total and the nuclear transfer protein domain. The upper sequence is a human nuclear transfer protein 10 containing an ATP / GTP binding domain, and the lower sequence is a nuclear transfer protein domain. Ί "and": "indicate that the probability of different amino acids appearing at the same position between the two sequences decreases in sequence.

 FIG. 1 is a polyacrylamide gel electrophoresis image (SDS-PAGE) of an isolated human ATP / GTP binding domain-containing nuclear conversion protein 10 (SDS-PAGE). LOkDa is the molecular weight of the protein. The arrow indicates the isolated protein band. Realization Best Mode of 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. In the following examples, the experimental methods without specific conditions are usually performed according to conventional conditions, such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer Suggested conditions.

 Example 1: Cloning of human ATP / GTP binding domain-containing nuclear transfer protein 10

Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. Poly (A) raRNA ₀ 2ug poly (A) mRNA was isolated from total RNA using Quik raRNA Isolation Kit (Qiegene) to form cDNA by reverse transcription. The Smart cDNA cloning kit (purchased from Clontech) was used to insert the cDNA fragment into the multicloning site of pBSK (+) vector (Clontech) to transform DH5cc to form a cDNA library. Dye terminate cycle reaction 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 clone 0623F07 was new DNA. A series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions. The results show that the full-length cDNA of clone 0623F07 is 579bp (as shown in Seq ID N0: 1), and there is a 264bp open reading frame (0RF) from 206bp to 469bp, which encodes a new protein (such as Seq ID NO : Shown in 2). We named this clone PBS-0623F07, and the encoded protein was named human ATP / GTP binding domain-containing nuclear conversion protein 10.

 Example 2: Cloning of the gene encoding human ATP / GTP binding domain-containing nuclear transfer protein 10 by RT-PCR method. Using fetal brain cell total RNA as a template and oligo-dT as a primer for reverse transcription reaction to synthesize cDNA. Qiagene After purification of the kit, PCR amplification was performed with the following primers:

 Priraerl: 5'-ACGGCTGCGAGAAGACGAAGCTTAG-3 '(SEQ ID NO: 3)

 Primer2: 5'- TCATTCAACAAATGTTTATTAATAG- 3, (SEQ ID NO: 4)

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

 Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.

Amplification reaction conditions: reaction volume containing 50 μ 1 of ^{5 0mmol / L KC1, 10mmol /} L Tris- CI, (pH8.5), 1.5mmol / L MgCl 2, 200 μ mol / L dNTP, lOpmol primer , 1U of Taq DNA polymerase (Clontech). The reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94. C 30sec; 55 ° C 30sec; 72 ° C 2min „At the same time, set β-act in as a positive control and template blank as a negative control at the time of RT-PCR. Amplification products were purified using QIAGEN's kit and TA cloning kit Linked to the pCR vector (Unvitrogen product). DNA sequence analysis results show that the DNA sequence of the PCR product is exactly the same as the 1- 579bp shown in SEQ II) NO: 1.

 Example 3: Northern blot analysis of human ATP / GTP binding domain-containing nuclear transfer protein 10 gene expression:

Total RNA was extracted by a one-step method [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 time volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ) And 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. Use 20 g RNA in 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-¼ M sodium acetate -Perform electrophoresis on a 1.2% agarose gel of ImMEDTA- 2.2M formaldehyde. It was then transferred to a nitrocellulose membrane. ³² P dATP labeled probe ³² P- DM prepared by the random primer Method - with α. The DNA probe used was the human ATP / GTP binding crust domain-containing nuclear conversion protein 10 coding region sequence (206p to 469bp) amplified by PCR shown in FIG. A 32P-labeled probe (approximately 2 10 ° cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH ₂ P0 ₄ ( pH7.4) -5 x SSC-5 x Denhardt's solution and 200 μg / ml salmon sperm DN'A. After hybridization, the filters were washed in 1 x SSC-0.1% SDS at 55 C for 30 min. Then, Phosphor Imager was used for analysis and determination Example 4: Recombinant human ATP / GTP binding domain-containing nuclear conversion protein 10 in vitro expression, isolation and purification According to the sequence of the coding region shown in SEQ ID NO: 1 and Figure 1, A pair of specific amplification primers was designed with the following sequence: Primer3: 5'- CCCCATATGATGCTGCGAACAGCTGGAGGGCAGG- 3 '(Seq ID No: 5)

 Primer4: 5'- CCCGAATTCTCAGAGACAATTACCATACACTACA- 3 '(Seq ID No: 6)

 The 5 'ends of these two primers contain Ndel and EcoRI restriction sites, respectively, followed by the coding sequences of the 5' and 3 'ends of the target gene, respectively. The Nde I and EcoR I restriction sites correspond to the expression vector plasmid ET- 28 b (+) (Novagen, Cat. No. 69865.3) a selective endonuclease site. PCR was performed using the pBS-0623F07 plasmid containing the full-length target gene as a template. The PCR reaction conditions were as follows: a total volume of 50μ1 containing 10 pg of pBS-0623F07 plasmid, Primer-3 and Primer-4 were lOpmol Advantage polymerase Mix (Clontech) 1 μ1, respectively. Cycle parameters: 94 C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles. Ndel and EcoRI were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into colibacillus DH5a by calcium chloride method. After being cultured overnight in LB plates containing kanamycin (final concentration 30yg / ml), positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0623F07) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE 3) p 1 y S s (product of Novagen) by the calcium chloride method. In LB liquid medium containing kanamycin (final concentration 30 μg / ral), the host bacteria BL21 (pET-0623F07) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1 mmol / L, and continued Incubate for 5 hours. The bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. The affinity chromatography column His. Bind Quick Cartridge (product of Novagen) was used to obtain 6 histidine (6His-Tag). The purified ATP / GTP binding domain-containing nuclear conversion protein 10 was purified by SDS-PAGE electrophoresis, and a single band was obtained at 10 kDa (Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by Edams hydrolysis method. As a result, the 15 amino acids at the N-terminus were identical to the 15 amino acid residues at the N-terminus shown in SEQ ID NO: 2.

 Example 5 Production of Anti-Human ATP / GTP Binding Domain-Containing Nuclear Conversion Protein 10 Antibodies

Synthesis of the following human ATP / GTP binding domain-containing nuclear conversion protein 10 using a peptide synthesizer (product of PE) Sexual peptides:

_{NH 2 -Met-Leu-Arg-} Thr-Ala-Gly-Gly-Gln-Va l-Met-Ser-Lys-Glu-Lys-Gl y-COOH (SEQ ID NO: 7) 0 , respectively, and the polypeptide Blood Coupling of cyanin and bovine serum albumin to form a complex, see Avrameas, et al. Immunochemi s try, 1969; 6: 43. Rabbits were immunized with 4 mg of the hemocyanin-polypeptide complex with complete Freund's adjuvant, and 15 days later, the hemocyanin-polypeptide complex plus incomplete Freund's adjuvant was used to boost the immunity once. 15Using a 15 μg / ml bovine serum albumin peptide complex-coated titer plate for ELISA to determine the antibody titer 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 demonstrated that the purified antibody could specifically bind to human ATP / GTP binding domain-containing nuclear conversion protein 10.

 Example 6: Application of the 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 tissues or 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 blotting, Northern blotting, and copying methods. They all use the same steps to hybridize the fixed polynucleotide sample to the filter. These same steps are as follows: The sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer so that the non-specific binding sites of the sample on the filter are saturated with the carrier and the synthetic 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) to reduce the hybridization background and retain only strong specific signals. 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 embodiment, 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 oligonucleotide fragment selected from the polynucleotide SEQ ID NO: 1 of the present invention for use as a hybridization probe should follow the following Principles and several aspects to consider:

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

 2, GC content is 30% -70%, non-specific hybridization increases when it exceeds;

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

 5 '-AAGGATCGAGGAAGGATGAAAATCGAGACAGAGAGAGAGAG-3' ^ SEQ ID NO: 9) For other commonly used reagents and their preparation methods related to the following specific experimental procedures, please refer to the literature: DNA PROBES GHKeller; MMManak; Stockton Press, 1989 (USA (USA) ) And more commonly used molecular cloning experiment manual books such as "Molecular Cloning Experiment Guide" (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) Suspend the pellet (approximately 10 ml / g) with a cold homogenization buffer (0.25 mol / L sucrose; 25 mmol / L Tris-HCl, pH 7.5; 25 cryptool / LnaCl; 25 surface 1 / L MgCl ₂ ). 4) at 4. C Use an electric homogenizer to homogenize the tissue suspension at full speed until the tissue is completely broken. 5) Centrifuge at 1000g for 0 minutes. 6) Resuspend the cell pellet (add 1-5 ml per 0.1 g of the original tissue sample) and centrifuge at 1,000 g for 10 minutes. 7) Resuspend the pellet with lysis buffer (add 1 ml per 0.1 g of the initial tissue sample), and then follow the phenol extraction method below.

2, phenol extraction method for DNA Steps: 1) Wash the cells with 1-10 ml of cold PBS and centrifuge at 1,000 g for 10 minutes. 2) with cold cell lysate pelleted cells were resuspended (1 ¹⁰⁸ cells / ml) Minimum application lOOul lysis buffer. 3) Add SDS to a final concentration of 1%. If SDS is added directly 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 or incubated gently shaking for 1 hour at 37 ^D 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 DNA-containing aqueous phase 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 evaporate the surface ethanol. 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 pipette while gradually increasing TE, mix until the DNA is fully dissolved, and add approximately 1 ul per 1-5 10 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 100u _g / ml, and incubate at 37 ° C for 30 minutes. 9) Add SDS and proteinase K, the final concentration is 0.5% and 100ug / ml. 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, re-extract with an equal volume of chloroform: isoamyl alcohol (24: 1), and centrifuge for 10 minutes. 12) Carefully remove the water phase, add 1/10 volume of 2mol / L sodium acetate and 2.5 volume of cold ethanol, mix and let stand 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 4x 2 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, in order to follow the experimental steps later The film was washed with high-strength conditions and strength conditions, respectively.

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

3) Place on filter paper infiltrated with 0.1 mol / L NaOH, 1.5 mol / L NaCl for 5 minutes (twice), dry and place in the infiltration Filter on 0.5 mol / L Tris-HCl (pH 7.0) and 3 mol / L NaCl for 5 minutes (twice) and dry. 4) Caught 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 (0.1 IOD / Ιθμ 1), add 2 μ IKinase buffer, 8-10 uCi y- 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 (ΒΒΒλ)

 4) Pass through a Sephadex G-50 column.

5) Before the ³² P-Probe is washed out, start collecting the first peak (can be monitored by Monitor).

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

 7) Monitor the amount of isotope with a liquid scintillator

8) After the collection solution of the first peak is combined, the ³² P-Probe (the second peak is free γ- ³² P-dATP) to be prepared. Pre-hybridization

Place the sample film in a plastic bag, add 3-10 mg of prehybridization solution (lOxDenhardt's; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA).), Seal the bag, and shake in a 68 ^D C water bath for 2 hours. .

 Cross

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

 Wash film:

 High-intensity washing film:

 1) Take out the hybridized sample membrane.

 2) 2xSSC, 0.1% SDS, wash at 40 ° C for 15 minutes (twice).

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

 4) 0.1xSSC, 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) 0.1xSSC, 0.1% SDS, wash at 37 ° C for 15 minutes (twice).

4) In 0.1xSSC, 0.1% SDS, wash at 40 ° C for 15 minutes (twice), and dry at room temperature. X-ray autoradiography:

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

 Experimental results:

 The hybridization experiments performed under low-intensity membrane washing conditions showed no significant difference in the radioactivity of the above four probe hybrid spots; while the hybridization experiments performed under low-intensity membrane washing conditions, the radioactive intensity of probe 1 was significantly stronger than The radioactivity of the other three probe hybridization spots. Therefore, the 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 development disorders of the reproductive system, tumors and cancers of related tissues, and the like.

 The first stage of mammalian spermatogenesis is the beginning of sperm cell development, and the second stage is the formation of a complete, transcriptionally inactivated non-nucleosome structure through fusion and aggregation of chromatin. This fusion process involves the conversion of proteins. These specific conversion proteins play an important regulatory role in the second stage of mature sperm formation. Abnormal expression of these proteins will cause individuals to fail to form normal, mature sperm, which will cause various related reproductive system diseases

[Freder ic CHIRAT, Ar let te MARTINAGE et al., Eur. J. Biochem. 1991, 198: 13-20] Nuclear transfer protein 1 is one of the sperm cell-specific proteins and contains a specific nuclear transfer protein 1 domain The abnormal expression of this domain will directly lead to the abnormal function of the protein during spermatogenesis, which will cause various related diseases of the reproductive system and disorders of embryonic development.

 It can be seen that the abnormal expression of the human ATP / GTP binding domain-containing nuclear conversion protein 10 in the present invention will cause various diseases, especially diseases of the reproductive system and disorders of embryonic development. These diseases include, but are not limited to:

 Reproductive system diseases: Testicular and epididymal inflammation, testicular tumors such as seminoma, embryonic cancer, teratoma, chorionic carcinoma, yolk sac tumor, testicular stromal cell tumor, epididymal tumor, etc.

Embryonic disorders: spina bifida, craniocerebral fissure, anencephaly, cerebral bulge, foramen deformity, Down syndrome, congenital hydrocephalus, aqueduct malformation, dwarfism of cartilage hypoplasia, dysplasia of the spine Disease, pseudochondral dysplasia, Langer-Giedion syndrome, funnel chest, gonad hypoplasia, congenital adrenal hyperplasia, urethral fissure, crypt, with short stature syndromes such as Conradi syndrome and Danbol t-Cl os s syndrome, congenital glaucoma or cataract, congenital lens abnormality, congenital small eyelid fissure, retinal dysplasia, congenital optic nerve atrophy, congenital sensorineural hearing loss, cleft palsy, teratosis, Wi ll iams syndrome, Alagi l le syndrome, Bayer II Syndrome, etc.

 The polypeptide of the present invention, as well as its antagonists, agonists and inhibitors, can be directly used in the treatment of diseases, for example, it can treat various diseases, especially diseases of the reproductive system and disorders of embryonic development.

 The invention also provides methods for screening compounds to identify agents that increase (agonist) or repress (antagonist human ATP / GTP binding domain-containing nuclear conversion protein 10). Agonists enhance human ATP / GTP binding domain-containing nuclear Transforming protein 10 stimulates biological functions such as cell proliferation, and antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers. For example, mammalian cells or human-expressing ATP / GTP-containing structures can be expressed in the presence of drugs A membrane preparation of nuclear transfer protein 10 of the domain was cultured with labeled human nuclear transfer protein 10 containing an ATP / GTP binding domain. The ability of the drug to increase or block this interaction was then determined.

 Antagonists of human ATP / GTP binding domain-containing nuclear transfer protein 10 include screened antibodies, compounds, receptor deletions, and the like. Antagonists of human ATP / GTP-binding domain-containing nucleoproteins 10 can bind to human ATP / GTP-binding domain-containing nucleoproteins 10 and eliminate their functions, or inhibit the production of the polypeptide, or with the polypeptide The active site binding prevents the polypeptide from performing biological functions.

 When screening compounds as antagonists, human ATP / GTP-binding domain-containing nuclear transfer protein 10 can be added to a bioanalytical assay. The effects of interactions between humans 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 human ATP / GTP binding domain-containing nucleoprotein 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, 10 human ATP / GTP binding domain-containing nuclear transduction proteins should 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 present invention also provides antibodies directed against human ATP / GTP binding domain-containing nuclear conversion protein 10 epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments generated from Fab expression libraries.

Polyclonal antibodies can be produced by injecting human ATP / GTP binding domain-containing nuclear transfer protein 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 it is not limited to Freund's adjuvant. Techniques for preparing human monoclonal antibodies containing ATP / GTP binding domain-containing nuclear transfer protein 10 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human beta- Cell hybridoma technology, EBV-hybridoma technology, etc. Human constant region and non-human variable region Bound chimeric antibodies can be produced using existing techniques (Morrson et al, PNAS, 1985, 81: 6851), and existing techniques for producing single-chain antibodies (0J. S. Pat No. 4946778) can also be used. Production of single-chain antibodies against human ATP / GTP binding domain-containing nuclear transfer protein 10.

 Antibodies against human ATP / GTP binding domain-containing nuclear transfer protein 10 can be used in immunohistochemical techniques to detect human ATP / GTP binding domain-containing nuclear transfer protein 10 in biopsy specimens.

 Monoclonal antibodies that bind to human ATP / GTP binding domain-containing nucleoprotein 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, human ATP / GTP binding domain-containing nuclear conversion proteins. 10 High-affinity monoclonal antibodies can covalently bind to bacterial or phytotoxins (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 humans that contain the ATP / GTP binding domain. Nuclear transfer protein 10 positive cells.

 The antibodies of the present invention can be used to treat or prevent diseases related to human ATP / GTP binding domain-containing nuclear conversion protein 10. Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human ATP / GTP binding domain-containing nuclear converter protein 10.

 The invention also relates to a diagnostic test method for the quantitative and localization detection of human ATP / GTP binding domain-containing nuclear converter protein 10 levels. These tests are well known in the art and include FISH assays and radioimmunoassays. The level of human ATP / GTP binding domain-containing nuclear transfer protein 10 detected in the test can be used to explain the importance of human ATP / GTP binding domain-containing nuclear transfer protein 10 in various diseases and for diagnosis Diseases in which human ATP / GTP binding domain-containing nucleoprotein 10 functions.

 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.

Polynucleotides encoding human ATP / GTP binding domain-containing nuclear conversion protein 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 human ATP / GTP binding domain-containing nuclear converter protein 10. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human ATP / GTP binding domain-containing nuclear conversion protein 10 to inhibit endogenous human ATP / GTP binding domain-containing nuclear conversion protein 10 activity. For example, a mutated human ATP / GTP binding domain-containing nuclear transfer protein 10 may be a shortened human ATP / GTP binding domain-containing nuclear transfer protein 10 that lacks a signaling domain. Substrate binding, but lacks signaling activity. Therefore heavy The group of gene therapy vectors can be used for treating diseases caused by abnormal expression or activity of nuclear conversion protein 10 containing human ATP / GTP binding domain. Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding a human ATP / GTP binding domain-containing nuclear conversion protein 10 to a cell Inside. Methods for constructing a recombinant viral vector carrying a polynucleotide encoding a human ATP / GTP binding domain-containing nuclear transfer protein 10 can be found in the literature (Sambrook, et al.). In addition, a recombinant polynucleotide encoding human ATP / GTP binding domain-containing nuclear transfer protein 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.

 ^ Human oligonucleotides (including antisense RNA and DNA) containing ATP / GTP binding domain nucleoprotein 10 mRNA and ribozymes are also within the scope of the present invention. A ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation. Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technology, such as solid-phase phosphate amide chemical synthesis to synthesize oligonucleotides. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA 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.

Polynucleotides encoding human ATP / GTP binding domain-containing nuclear conversion protein 10 can be used for the diagnosis of diseases related to human ATP / GTP binding domain-containing nuclear conversion protein 10. Polynucleotides encoding human ATP / GTP binding domain-containing nucleoproteins 10 can be used to detect the expression of human ATP / GTP binding domain-containing nucleoproteins 10 or human ATP / GTP binding structures in disease states Domain of Aberrant Expression of Nuclear Conversion Protein 10 For example, a DNA sequence encoding a human ATP / GTP binding domain-containing nuclear transfer protein 10 can be used to hybridize biopsy specimens to determine the expression of human ATP / GTP binding domain-containing nuclear transfer protein 10. Hybridization techniques include Southern blotting, Northern blotting, and in situ hybridization. These techniques and methods are publicly available mature techniques, and related kits are commercially available. Some or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissue. Human ATP / GTP binding domain-containing nucleoprotein 10 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect human ATP / GTP binding domain-containing nucleoprotein 10 transcripts . Detection of mutations in the human ATP / GTP-binding domain-containing nuclear transfer protein 10 gene can also be used to diagnose human ATP / GTP-binding domain-containing nuclear transfer protein 10-related diseases. Human ATP / GTP binding domain-containing nuclear conversion protein 10 mutant forms include point mutations, translocations, deletions, recombinations, and others compared to normal wild-type human ATP / GTP binding domain-containing nuclear conversion protein 10 DNA sequences Any exceptions etc. Mutations can be detected using existing techniques such as Southern blotting, DNA 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. This sequence will specifically target a specific position of a human chromosome and can hybridize with 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) can be used to mark 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 based on cDNA, and the sequences can be mapped on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells containing human genes corresponding to the primers 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, sublocalization can be achieved using a set of fragments from a specific chromosome or a large number of genomic clones. Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection 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 Manual of Basic Techniques, Pergamon Press, 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 Welch 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 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, they are accurately mapped to disease-related chromosomal regions. cDNA can be one of between 50 and 500 potentially pathogenic genes (assuming 1 megabase mapping resolution and one gene per 20 kb).

 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 contains a safe and effective amount of the polypeptide or antagonist, and carriers and excipients that 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 manufacture, use, or sell them. 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. Human ATP / GTP binding domain-containing nuclear conversion protein 10 is administered in an amount effective to treat and / or prevent a specific indication. The amount and dose range of human ATP / GTP binding domain-containing nuclear conversion protein 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

1. An isolated polypeptide-human ATP / GTP binding domain-containing nuclear conversion protein 10, characterized in that it comprises: a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, or an active fragment of the polypeptide, similar Thing or derivative. 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, further comprising 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 an amino acid sequence shown in SEQ ID NO: 2 or a fragment, analog, or derivative thereof;  (b) a polynucleotide complementary to the polynucleotide (a); or  (c) a polynucleotide that is at least 70% identical to) or (b).  5. The polynucleotide according to claim 4, characterized in that said 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 206-469 in SEQ ID NO: 1 or the sequence of positions 1-577 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 a polynucleotide according to any one of claims 4-6.  9. A method for preparing a polypeptide having human ATP / GTP binding domain-containing nuclear transfer protein 10 activity, characterized in that the method comprises:  (a) culturing the engineered host cell of claim 8 under the condition of expressing a human ATP / GTP binding domain-containing nuclear transfer protein 10;  (b) Isolating a polypeptide having human ATP / GTP binding domain-containing nucleoprotein 10 activity from the culture. 10. An antibody capable of binding to a polypeptide, characterized in that said antibody is an antibody capable of specifically binding to a human ATP / GTP binding domain-containing nuclear conversion protein 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 a human nuclear transfer protein 10 containing an ATP / GTP binding domain.  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 a human ATP / GTP binding domain-containing nuclear conversion protein 10 in vitro and in vivo.  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 human mimetics, agonists, antagonists or antagonists of nuclear conversion protein 10 containing an ATP / GTP binding domain. Inhibitors; or for peptide fingerprinting.  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 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 human ATP / GTP binding domain-containing nuclear conversion protein 10 abnormalities.  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.