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WO2001072800A1 - Nouveau polypeptide, proteine humaine d'echange nucleotidique 13 contenant un domaine de liaison atp/gtp, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, proteine humaine d'echange nucleotidique 13 contenant un domaine de liaison atp/gtp, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001072800A1
WO2001072800A1 PCT/CN2001/000437 CN0100437W WO0172800A1 WO 2001072800 A1 WO2001072800 A1 WO 2001072800A1 CN 0100437 W CN0100437 W CN 0100437W WO 0172800 A1 WO0172800 A1 WO 0172800A1
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Prior art keywords
polypeptide
polynucleotide
binding domain
protein
gtp binding
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc
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Shanghai Biowindow Gene Development Inc
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Priority to AU56074/01A priority Critical patent/AU5607401A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, a human nuclear transfer protein 1 3 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.
  • Sperm formation is a process in which a gamete is gradually differentiated and finally mature sperm cells are formed.
  • 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.
  • TP1, TP2, TP3, and TP4 four transforming proteins have been cloned from rats and mice. The study of TP1 and TP2 is more detailed. Subsequently, human and bovine TP1 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 is composed 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-KR-K- ⁇ -RK.
  • the sequence fragment contains one 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-DNA interaction. Instability is required; and the phosphorylation of the serine site is also required for protein-DNA interactions, which may regulate the correct binding of the protein to specific sites in the DNA.
  • ATP or GTP binding motif is a ubiquitous domain in various ATP / GTP binding proteins. Some ATP or GTP-linked proteins contain this conservative raotif sequence more or less. This motif has a conservative characteristic sequence. Fragment: [AG] -X (4) -GK- [ST] (where X is any amino acid residue). The most conservative of these motifs is the glycine-rich region, which forms a flexible loop between the beta line and the alpha helix.
  • This curved loop usually reacts with a phosphate group on the nucleotide, and the motif sequence is often called the "A" consensus sequence or P-loop.
  • This motif is usually responsible for binding with ATP and GTP in the body, providing the energy required for the protein to perform biological functions and stabilizing the structure of the protein in the body, assisting the protein to complete its normal physiological function. Its 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 expression profile of the polypeptide of the present invention is very similar to the expression profile of human ATP / GTP binding domain-containing nuclear conversion protein 10, so the functions of the two may also be similar.
  • the present invention is named a human ATP / GTP binding domain-containing nuclear conversion protein 13.
  • the human ATP / GTP binding domain-containing nuclear transfer protein 13 protein plays an important role in regulating important functions of the body such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes. More human ATP / GTP binding domain-containing nuclear transfer protein 13 proteins need to be identified, especially the amino acid sequence of this protein. Isolation of the ATP / GTP binding domain-containing nuclear conversion protein 13 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 it is important to isolate its coding for DM. Object of the invention
  • 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 13.
  • Another object of the present invention is to provide a genetically engineered host cell comprising a polynucleotide encoding a human ATP / GTP binding domain-containing nuclear transduction protein 13.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors of the polypeptide of the present invention, human ATP / GTP binding domain-containing nuclear conversion protein 13.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of human nuclear transfer protein 13 containing an ATP / GTP binding domain.
  • 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.
  • 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:
  • polynucleotide sequences of (c) and (a) or (b) have at least 70 ° /. Identical polynucleotides.
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 173-526 in SEQ ID NO: 1; and (b) having a sequence of 1-1 in SEQ ID NO: 1 5 36-bit sequence.
  • the present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; 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 converter protein 13 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 associated with abnormal expression of a human ATP / GTP binding domain-containing nuclear transfer protein 13 protein, 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 invention also relates to a pharmaceutical composition
  • 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 expression of the polypeptides and / or polynucleotides of the present invention in the preparation of nuclear conversion protein 1 3 for use in the treatment of cancer, developmental or immune diseases, or other human-derived ATP / GTP binding domains. Use of medicines for diseases caused by abnormalities.
  • Fig. 1 is a comparison diagram of gene chip expression profiles of the present inventor's ATP / GTP binding domain-containing nuclear conversion protein 13 and human ATP / GTP binding domain-containing nuclear conversion protein 103.
  • the upper figure is a graph of human ATP / GTP binding domain-containing nuclear conversion protein 1 3, and the lower figure is the human ATP / GTP binding domain-containing nuclear protein 10 expression profile.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of an isolated human ATP / GTP-binding crust domain-containing nuclear conversion protein 1 3.
  • 1 3kDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • 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 “variant" of a protein or polynucleotide 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 amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine 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 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 a different amino acid Or nucleotides replace one or more amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind 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 13, 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 transduction protein 13.
  • Antagonist refers to a nuclear conversion protein that blocks or regulates a human ATP / GTP binding domain-containing 1 when it binds to a human ATP / GTP binding domain-containing nuclear conversion protein 1 3
  • 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 13.
  • Regular refers to a change in the function of a human ATP / GTP binding domain-containing nuclear transfer protein, including an increase or decrease in protein activity, a change in binding characteristics, and a human ATP / GTP binding domain-containing nuclear conversion protein. 1 3 any other biological, functional or immune change.
  • Substantially pure means substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. Those skilled in the art can purify human nuclear conversion proteins containing ATP / GTP binding domains using standard protein purification techniques. Substantially pure human ATP / GTP binding domain-containing nuclear transfer proteins 1 3 can generate a single main band on a non-reducing polyacrylamide gel. The purity of the human ATP / GTP binding domain-containing nuclear transfer protein 1 3 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence C-T-G-A
  • 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. This inhibition of hybridization can be detected by performing hybridization (Sou the rn imprint or Nor thern blot, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully 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 means the sequence is the same in two or more amino acid or nucleic acid sequence comparisons Similar percentages.
  • 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 according to different methods such as the Clus ter method (Hi gg ins, DG and PM Sharp (1988) Gene 73: 237-244). 0
  • the Clus ter method checks the distance between all pairs.
  • the groups of sequences are arranged into 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:
  • 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 in enzymology 183: 625-645). 0
  • 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 substitution 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 HFP or a chemical modification of its nucleic acid. 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,? ( ⁇ ') 2 and? It can specifically bind to human epitopes of nuclear conversion protein 13 containing an ATP / GTP binding domain.
  • 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.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, 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 part of its natural environment, they are still isolated.
  • 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).
  • Polynucleoside in its natural state Acids and polypeptides are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances in their natural state.
  • isolated human ATP / GTP binding domain-containing nuclear conversion protein 1 3 refers to human ATP / GTP binding domain-containing nuclear conversion protein 1 3 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 1 3 using standard protein purification techniques. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the human ATP / GTP binding domain-containing nuclear transfer protein 1 3 peptide can be analyzed by amino acid sequence.
  • the present invention provides a novel polypeptide, a human ATP / GTP binding domain-containing nuclear conversion protein 1 3, 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 invention may be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells).
  • polypeptides of the invention may be glycosylated, or they may be non-glycosylated.
  • the polypeptides of the invention may also include or exclude the initial methionine residue.
  • the invention also includes fragments, derivatives, and analogs of human ATP / GTP binding domain-containing nuclear conversion proteins 1 3.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human ATP / GTP binding domain-containing nuclear conversion protein 1 3 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 (II) a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or ( ⁇ ⁇ ) Such a polypeptide sequence in which the mature polypeptide is fused with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV) a polypeptide sequence in which an additional amino acid sequence is fused into the mature polypeptide (Such as the leader or secretory sequence or the sequence used to purify the polypeptide or protease sequence). 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.
  • Polynucleotides of the invention are found from a CDM library of human fetal brain tissue. It contains a full-length polynucleotide sequence of 1536 bases and its open reading frames 173-526 encode 117 amino acids.
  • the conversion protein 1 0 has a similar expression profile, and it can be deduced that the human ATP / GTP binding domain-containing nuclear conversion protein 1 3 has similar functions as the human ATP / GTP binding domain-containing nuclear conversion protein 10.
  • 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.
  • DNA can be coding or non-coding.
  • 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.
  • 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.
  • 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.
  • 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 present 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.
  • “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% F i co ll, 42 ° C, etc .; or (3) only between the two sequences Crosses occur at least 95% or more, and more preferably 97% or more.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, most preferably at least 100 More than nucleotides.
  • 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 nuclear conversion protein 1 3.
  • the polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • polynucleotide sequence of the present invention encoding human ATP / GTP binding domain-containing nuclear transfer protein 13 can be obtained by various methods.
  • 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 the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • 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.
  • mRNA extraction There are many mature techniques for mRNA extraction, and kits are also commercially available (Qiagene :).
  • the 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 cDM 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): (1) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of human ATP / GTP binding domain-containing nuclear transfer protein 13 The level of transcripts; (4) Detecting protein products expressed by genes by immunological techniques or by measuring biological activity. The above methods can be used alone or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and has a length of at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • 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.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of human ATP / GTP-binding domain-containing nuclear conversion protein 13 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). Wait.
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). Wait.
  • a method of amplifying DNA / RNA by PCR is preferably used to obtain the gene of the present invention. Especially difficult to get from the library
  • the RACE method RACE-rapid cDNA end rapid amplification method
  • the primers used for PCR can be appropriately selected according to the polynucleotide sequence information of the present invention disclosed herein, and conventional methods can be used. synthesis.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • 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. In order to obtain the full-length cDNA sequence, sequencing needs to 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 13 coding sequence, and produced by recombinant technology A method of a polypeptide according to the invention.
  • a polynucleotide sequence encoding a human ATP / GTP binding domain-containing nuclear conversion protein 13 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • 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 expressed in bacteria (Rosenberg, et al.
  • any plasmid and vector can be used to construct recombinant expression vectors.
  • 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 human ATP / GTP binding domain-containing nuclear transfer protein 13 and appropriate 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. Representative examples of these promoters are: the lac or trp promoter of E.
  • 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 expressed by DM. There are usually about 10 to 300 base pairs that 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 and adenovirus enhancers on the late side of the origin of replication.
  • 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.
  • 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.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding a human ATP / GTP binding domain-containing nuclear conversion protein 1 3 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute the polynucleotide or the recombinant vector.
  • Genetically engineered host cells 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
  • fly S2 or Sf 9 animal cells
  • animal cells such as CH0, COS or Bowes s melanoma cells, etc. .
  • 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.
  • the host is a prokaryote such as E. coli
  • competent cells capable of DNA uptake can be harvested after exponential growth phase, with ( ⁇ (1 2 Treatment, procedure used are well known in the art. Alternatively, it is used MgC l 2.
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and lipid Plastid packaging, etc.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human ATP / GTP binding domain-containing nuclear conversion protein 1 (Scence, 1984; 224: 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. When the host cell has 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.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its 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.
  • 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
  • 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.
  • the first stage of mammalian spermatogenesis is the beginning of sperm cell development, and the second stage is to form 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 l et te MARTINAGE et a l., Eur. J. Biochem.
  • 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.
  • the abnormal expression of the human ATP / GTP binding domain-containing nuclear conversion protein 13 in the present invention will produce various diseases, especially diseases of the reproductive system and disorders of embryonic development. These diseases include, but are not limited to:
  • 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 spinal epiphysis Disease, pseudochondral dysplasia, Langer-Gie ed i on syndrome, funnel chest, gonad hypoplasia, congenital adrenal hyperplasia, urethral fissure, cryptorchidism, short stature syndromes such as Conradi syndrome and Danbo l t-Cl os s syndrome, congenital glaucoma or cataract, congenital lens position abnormality, congenital blepharoplasia, retinal dysplasia, congenital optic nerve atrophy, congenital sensorineural hearing loss, cracked hands and cracked feet, Teratosis, Wi ll iams syndrome, Al ag ille syndrome, Bay
  • the invention also provides screening compounds to identify human compounds that increase (agonist) or suppress (antagonist)
  • Method of ATP / GTP-binding Domain of Nuclear Conversion Protein 1 3 Agonists enhance human ATP / GTP binding domain-containing nuclear conversion proteins. 13 Stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • a mammalian cell or a membrane preparation expressing a human ATP / GTP binding domain-containing nuclear conversion protein 1 3 can be labeled with a labeled human ATP / GTP binding domain-containing nuclear conversion protein 1 3 in the presence of a drug. From cultivation. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human ATP / GTP binding domain-containing nuclear transfer proteins 13 include screened antibodies, compounds, receptor deletions, and the like. Antagonists of human ATP / GTP binding domain-containing nuclear transfer protein 13 can bind to human ATP / GTP binding domain-containing nuclear conversion protein 13 and eliminate its function, or inhibit the production of the polypeptide, or The active site binding of the polypeptide prevents the polypeptide from performing a biological function.
  • human ATP / GTP binding domain-containing nuclear conversion protein 1 3 can be added to the bioanalytical assay, and the human ATP / GTP binding domain-containing nuclear conversion protein 1 3 and The effect of their receptor interactions to determine whether a compound is an antagonist.
  • Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to human ATP / GTP binding domain-containing nuclear conversion protein 1 3 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. During screening, human ATP / GTP binding domain-containing nuclear transfer protein 13 molecules should generally be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the present invention also provides antibodies against human ATP / GTP binding domain-containing nuclear conversion protein 13 epitopes. 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 human ATP / GTP binding domain-containing nuclear transfer protein 1 3 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • 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 and the like.
  • Techniques for preparing human monoclonal antibodies containing ATP / GTP binding domain-containing nuclear transfer protein 13 include, but are not limited to, hybridoma technology (Koh ler and Milstei n. Nature, 1975, 256: 495-497), three tumors Technology, human B-cell hybridoma technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions and non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851).
  • the existing technology for producing single chain antibodies (US Pat No. 4946778) can also be used to produce single chain antibodies against human ATP / GTP binding domain-containing nuclear conversion protein 13.
  • Antibodies against human ATP / GTP binding domain-containing nuclear conversion protein 13 antibodies can be used in immunohistochemistry to detect human ATP / GTP binding domain-containing nuclear conversion protein 13 in biopsy specimens.
  • Monoclonal antibodies that bind to human ATP / GTP binding domain-containing nuclear transfer protein 13 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.
  • 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 human ATP / GTP binding domain-containing Nuclear transfer protein 13 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 transduction protein 13.
  • 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 13.
  • 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 13 levels.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • Human ATP / GTP binding domain-containing nuclear transfer protein 13 levels detected in the test can be used to explain the importance of human ATP / GTP binding domain-containing nuclear transfer protein 13 in various diseases and to diagnose humans Diseases in which ATP / GTP-binding domain-containing nuclear converter protein 13 functions.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • 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 13 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 13.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human ATP / GTP binding domain-containing nuclear conversion protein 13 to inhibit endogenous human ATP / GTP binding domain-containing nuclear conversion protein 13 active.
  • a mutated human ATP / GTP binding domain-containing nuclear transfer protein 13 may be a shortened human ATP / GTP binding domain-containing nuclear transfer protein 13 that lacks a signaling domain, although it may be associated with downstream Substrate binding, but lacks signaling activity. Therefore, recombinant gene therapy vectors can be used for treatment W 01/7 Treats diseases caused by abnormal expression or activity of nuclear conversion protein 1 3 with ATP / GTP binding domain.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc.
  • a polynucleotide encoding a human ATP / GTP binding domain-containing nuclear conversion protein 1 3 can be used to transfer a polynucleotide encoding a human ATP / GTP binding domain-containing nuclear conversion protein 1 3 to in the cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a human ATP / GTP binding domain-containing nuclear transfer protein 1 3 can be found in existing literature (Sambrook, eta l.).
  • recombinant polynucleotides encoding human ATP / GTP binding domain-containing nuclear transfer protein 1 3 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.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense R and DNA
  • ribozymes that inhibit human ATP / GTP binding domain-containing nuclear transfer protein 1 3 mRNA 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, DM, and ribozymes can be obtained using any existing RNA or DM 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 vector's RNA polymerase promoter. 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 thiophosphate or peptide bonds instead of phosphodiester bonds.
  • a polynucleotide encoding a human ATP / GTP binding domain-containing nuclear transfer protein 1 3 can be used for the diagnosis of diseases related to human ATP / GTP binding domain-containing nuclear conversion protein 13.
  • Polynucleotides encoding human ATP / GTP binding domain-containing nuclear transfer protein 1 3 can be used to detect the expression of human ATP / GTP binding domain-containing nuclear transfer protein 1 3 or humans containing ATP / GTP under disease conditions Abnormal expression of nuclear conversion protein 13 in the binding domain.
  • the DNA sequence encoding human ATP / GTP binding domain-containing nuclear transfer protein 1 3 can be used to hybridize biopsy specimens to determine the expression of human ATP / GTP binding domain-containing nuclear transfer protein 1 3.
  • Hybridization techniques include blotting Sou t hern t Nor t hern blotting, in situ hybridization. These techniques and methods are publicly available and mature, 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 (Mic Roa ray) or a DNA chip (also known as a "gene chip") for analyzing differential expression analysis of genes in tissues and Genetic diagnosis, RNA-polymerase chain reaction (RT-PCR) using human ATP / GTP binding domain-containing nuclear transfer protein 13 specific primers for in vitro amplification can also detect human ATP / GTP binding domain-containing nuclear conversion protein 1 of 3 transcripts.
  • Human ATP / GTP binding domain-containing nuclear transfer protein 13 mutant forms include point mutations, translocations, deletions, recombinations, and others compared to normal wild-type human ATP / GTP binding domain-containing nuclear conversion protein 13 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. Therefore, Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • sequences of the invention are also valuable for chromosome identification. This sequence will specifically target 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.
  • PCR primers (preferably 15-35bp) are prepared based on cDM, and the sequences can be mapped to hybrid cells stained with human genes of those corresponding primers to produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • 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 F ISH
  • F ISH Fluorescent in situ hybridization
  • 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 diseased and unaffected individuals usually involves first looking for structural changes in the chromosome, such as defects visible at the chromosomal level or detectable by cDNA sequence-based PCR Missing or transposing. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 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.
  • suitable pharmaceutical carrier 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.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • 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.
  • 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 nucleoprotein 13 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 13 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. Examples
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using Quik mRNA I solat ion Kit (product of Qiegene). 2ug poly (A) mRNA forms CDM by reverse transcription.
  • a Smar t cDNA cloning kit purchased from C 1 on t ech was used to insert the cDNA fragment into the multi-cloning site of the pBSK (+) vector (Clontech) to transform DH5 ⁇ to form a cDNA library.
  • Dye terminate cycle reaction ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public DM sequence database (Genebank), and one of the clones was found.
  • the 0825 ⁇ 2 cDNA sequence was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, the following primers were used for PCR amplification:
  • Primer2 5,-TTCTTGCACAAATATTTTATTCTT -3, (SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp at the 5 'end of SEQ ID NO: 1;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Conditions for the amplification reaction 50 mmol / L KC1, 10 mmol / L Tris-HCl, pH 8.5, 1.5 ramol / L MgCl 2 , 200 jimol / L dNTP, lOpmol primer, 1U Taq DNA polymerase in a 50 ⁇ 1 reaction volume (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.
  • ⁇ -actin was set as a positive control and template blank was set as a negative control.
  • This method involves acid guanidinium thiocyanate phenol-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 ), Mix and centrifuge. 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.
  • RNA With 20 ⁇ 8 RNA, electrophoresed on containing 20mM 3- (N- morpholino) propanesulfonic acid (PH7.0) 1.2% agarose gel Xiao -5raM -ImM EDTA-2.2M sodium acetate formaldehyde. It was then transferred to a nitrocellulose membrane.
  • the DM probe used was the nuclear transfection of a human ATP / GTP binding domain containing the PCR amplification shown in Figure 1. Sequence of the coding protein 13 coding region (173bp to 526bp).
  • a 32P-labeled probe (about 2 x 10 6 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 20 ( ⁇ g / ml salmon sperm DNA. After hybridization, the filter was washed in 1 x SSC-0.1% SDS at 55 ° C for 30 minutes. Then, use Phosphor Imager was used for analysis and quantification.
  • Example 4 In vitro expression, isolation and purification of recombinant human ATP / GTP binding domain-containing nuclear transfer protein 13
  • Primer3 5'- CCCCATATGATGATCATTGACAGCTCCCGCATC -3, (Seq ID No: 5)
  • Primer4 5,-CAT'GGATCCTCAGCCTTTGAGATAATTGTGAAA -3, (Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and BamHI digestion sites, respectively, followed by the 5' and 3 'ends of the target gene, respectively.
  • Sequence, Ndel and BamHI restriction sites correspond to selective endonuclease sites on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3).
  • a PCR reaction was performed using the PBS-0825 ⁇ 2 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of pBS-0825 ⁇ 2 plasmid, primers Primer-3 and Primer-4 were lOpraol, Advantage polymerase Mix (Clontech) 1 ⁇ 1, respectively. Cycle parameters: 94. C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles. Ndel and BamHI 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 E. coli DH5CX using the calcium chloride method.
  • the bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. The chromatography was performed using an affinity column His. Bind Quick Cartridge (product of Novagen) capable of binding to 6 histidines (6His-Tag). A purified nuclear conversion protein 13 containing the ATP / GTP binding domain of the target protein was obtained. After SDS-PAGE electrophoresis, a single band was obtained at 13 kDa ( Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by the Edams hydrolysis method.
  • 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 sera using protein A-Sepharose.
  • the peptide was bound to a cyanogen bromide-activated Seph ar 0 S e4B 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 human nuclear conversion protein 13 containing ATP / GTP binding domain.
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • 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.
  • 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.
  • Filter hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods. They all use the same steps of hybridization after fixing the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer, so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthetic polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment utilizes 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.
  • the dot blot method is used to fix the sample on the filter membrane. Needle-to-sample hybridization has the strongest specificity and is retained.
  • 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:
  • the preferred range of probe size is 18-50 nucleotides
  • 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 For homology comparison of the regions, 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 generally;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 1 which belongs to the second type of probe, is equivalent to the replacement mutation sequence (41Nt) of the gene fragment or its complementary fragment of SEQ ID NO: 1:
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • the sample membrane was placed in a plastic bag, and 3-10 mg of prehybridization solution (10xDenhardt's; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA)) was added. After sealing the mouth of the bag, shake at 68 ° C for 2 hours.
  • prehybridization solution 10xDenhardt's; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA)
  • Gene chip or gene micro-matrix is a new technology that many national laboratories and large pharmaceutical companies are developing and developing. , Silicon and other carriers, and then use fluorescence detection and computer software to compare and analyze the data, in order to achieve the purpose of rapid, efficient, high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • the specific method steps have been reported in the literature. For example, see DeR isi, L L., Lyer, V. & Brown, P. 0. (1997) Sc ience 278, 680- 686. and He lle, RA, Schema, M., Cha i, A., Sha lom, D., (1997) PNAS 94: 2150-2155.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were respectively amplified by PCR, and the concentration of the amplified product was adjusted to about 500ng / ul after purification. The spots were spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 ⁇ . The spotted slides were hydrated, dried, and cross-linked in a UV cross-linking instrument. After elution, the DNA was fixed on the glass slide to prepare a chip. The specific method steps have been variously reported in the literature. The post-spotting processing steps of this embodiment are:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and mRNA was purified using Oligotex raRNA Midi Kit (purchased from QiaGen).
  • the fluorescent reagent Cy3dUTP 5-Amino-propargyl-2'-deoxyuridine 5--triphate coupled to Cy3 fluorescent dye (purchased from Araersham Phamacia Biotech) was used to label the mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP (5- Amino-propargyl- 2'-deoxyuridine 5 '-triphate coupled to Cy5 fluorescent dye, purchased from Amershara Phamacia Biotech company, labeled the body's specific tissue (or stimulated cell line) mRNA, and purified the probe to prepare a probe.
  • Cy3dUTP 5-Amino-propargyl-2'-deoxyuridine 5--triphate coupled to Cy3 fluorescent dye (purchased from Araersham Phamacia Biotech)
  • Probes from the two types of tissues and the chips were hybridized in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, washed with a washing solution (1> ⁇ SSC, 0.2% SDS) at room temperature, and then scanned with ScanArray.
  • the 3000 scanner purchased from General Scanning, USA was used for scanning. The scanned images were analyzed and processed with Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, Arsenic stimulated the L02 cell line and prostate tissue for 1 hour. Based on these 13 Cy3 / Cy5 ratios, draw a bar graph ( Figure 1). It can be seen from the figure that the expression profile of human ATP / GTP binding domain-containing nuclear conversion protein 13 and human ATP / GTP binding domain-containing nuclear conversion protein 10 are very similar.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine humaine d'échange nucléotidique 13 contenant un domaine de liaison ATP/GTP, et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la protéine humaine d'échange nucléotidique 13 contenant un domaine de liaison ATP/GTP.
PCT/CN2001/000437 2000-03-27 2001-03-26 Nouveau polypeptide, proteine humaine d'echange nucleotidique 13 contenant un domaine de liaison atp/gtp, et polynucleotide codant pour ce polypeptide Ceased WO2001072800A1 (fr)

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CN00115183A CN1315394A (zh) 2000-03-27 2000-03-27 一种新的多肽——人含atp/gtp结合结构域的核转换蛋白13和编码这种多肽的多核苷酸
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Non-Patent Citations (3)

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Title
DATABASE GENBANK [online] 23 November 1999 (1999-11-23), "Human DNA sequence from clone 272L16 on chromosome 1q32.1-32. 3. Contains the 3' end of the LAMB3 gene for laminin, beta 3(Nicein, kalinin, BM600) and a novel rat Ca2+/calmodulin dependent protein kinase LIKE gene. Contains ESTs, STSs, GSSs, genomic marker D1S491", Database accession no. AL023754 *
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KAWATA M. ET AL.: "The molecular heterogeneity of the smg-21/Krev-1/rap1 proteins, a GTP-binding protein having the same effector domains as ras p21s, in bovine aortic smooth muscle membranes", ONCOGENE, vol. 6, no. 5, 1991, pages 841 - 848 *

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AU5607401A (en) 2001-10-08

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