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WO2002020588A1 - Nouveau polypeptide, isomere humain zfm1 25.63 du facteur de regulation et de transcription, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, isomere humain zfm1 25.63 du facteur de regulation et de transcription, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002020588A1
WO2002020588A1 PCT/CN2001/001127 CN0101127W WO0220588A1 WO 2002020588 A1 WO2002020588 A1 WO 2002020588A1 CN 0101127 W CN0101127 W CN 0101127W WO 0220588 A1 WO0220588 A1 WO 0220588A1
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Prior art keywords
polypeptide
polynucleotide
zfm1
isomer
human transcriptional
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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 AU2002223372A priority Critical patent/AU2002223372A1/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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • 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
    • 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 transcriptional regulatory factor ZFM1 isomer 25. 63, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique
  • transcriptional regulation of eukaryotic genes is very important for the normal expression of genes, and this regulation process is usually completed by transcriptional regulatory factors.
  • Transcriptional regulators are involved in the body to determine in which tissues and developmental stages genes begin transcription. If a gene encoding such a protein is mutated, not only the gene itself cannot be expressed normally, but also many genes regulated by it cannot be normally transcribed and expressed. There are a large number of different types of transcriptional regulatory factors in higher organisms. These regulatory factors, in combination with various genetic materials DM and RM, regulate the transcription and expression of related genetic materials under the synergistic action of other related proteins.
  • transcription regulation factors People have cloned a variety of different transcription regulation factors from various organisms, such as various members of the zinc finger protein family. In addition to the conserved DM-binding domain, the protein sequences of these transcription factors also contain transcription activation regions. Transcriptional activation regions are mainly acidic amino acid types, glutamine-rich and proline-rich. These transcriptional activation regions interact with components of a universal transcriptional mechanism to regulate the transcription and expression of genes in the body. In 1998, Di Zhang et al. Cloned a new human transcriptional regulatory factor ZFM1 protein isoform B3 from humans, which has similar structural characteristics to the aforementioned transcription factors.
  • the protein has transcriptional repressive activity in vivo, which may work synergistically with some related transcription-activating proteins to control the transcription and expression of related proteins in vivo.
  • the N-terminus of the amino acid sequence of the protein contains a transcription suppression domain composed of about 137 amino acid residues, which is an important domain for the protein to exert transcription suppression activity. Mutations or abnormal expression of this domain will directly affect the protein's function in vivo, and then affect the transcription and expression of related proteins in vivo.
  • the protein plays an extremely important role in the body, and it is related to the expression of the MEN1 gene, which regulates the differentiation of tumor cells in the body to control the occurrence of malignant diseases such as tumors. Its mutation or abnormal expression is usually closely related to the occurrence of some endocrine tumors and nervous system diseases in the organism [Di Zhang, Geoffrey Chi Ids, 1998, J Bi ol Chem, 273: 6868-6877].
  • the ZFM1 isoform protein plays an important role in the transcription and expression of organism genes Regulatory effect, which works synergistically with some related transcriptional activating proteins to regulate the normal transcription and expression of proteins in vivo.
  • the mutation or abnormal expression of this protein will cause abnormal expression of related proteins in the body and abnormal proliferation of related cells, and then cause various related metabolic and developmental diseases.
  • the protein is usually closely related to the occurrence of some related metabolic and developmental disorders, nervous system diseases, endocrine disorders and related tissue tumors and cancers in the body. It can also be used to diagnose and treat the various related diseases mentioned above.
  • the 25.63 protein of the human transcriptional regulator ZFM1 isoform 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, so identification in the art has been required More human transcriptional regulators ZFM1 isomer 25. 63 protein involved in these processes, especially the amino acid sequence of this protein was identified. New human transcriptional regulator ZFM1 isomer 25. 63 The isolation of the protein-coding gene also provides a basis for research to determine the role of the protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic agents for diseases, so it is important to isolate its coding for DM. Disclosure of 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 the human transcriptional regulator ZFM1 isomer 25.63.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding the human transcriptional regulator ZFM1 isomer 25.63.
  • Another object of the present invention is to provide a method for producing the human transcriptional regulator ZFM1 isomer 25. 63.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention, a human transcriptional regulator ZFM1 isomer 25.63.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention-human transcriptional regulator ZFM1 isomer 25.63.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to the abnormality of the human transcriptional regulator ZFM1 isomer 25.63.
  • 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:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 1178-1879 in SEQ ID NO: 1; and (b) a sequence having 1-1989 in SEQ ID NO: 1 Sequence of bits.
  • 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 invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit human transcriptional regulator ZFM1 isomer 25.63 protein activity, which comprises using the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for detecting a disease or disease susceptibility related to the abnormal expression of the human transcriptional regulatory factor ZFM1 isomer 25.63 protein in vitro, which comprises detecting the polypeptide in the biological sample or its coding polynucleotide sequence Mutates, or detects the amount or biological activity of a polypeptide of the invention in a biological sample.
  • 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 preparation of a polypeptide and / or polynucleotide of the present invention for the treatment of cancer, developmental disease or immune disease or other drugs caused by abnormal expression of the human transcriptional regulator ZFM1 isomer 25.63. use.
  • 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 means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • 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 the human transcriptional regulator ZFM1 isomer 25.63, causes a change in the protein to regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to the human transcriptional regulator ZFM1 isomer 25.63.
  • Antagonist refers to a biological activity or immunity that can block or regulate the human transcriptional regulator ZFM1 isomer 25.63 when combined with the human transcriptional regulator ZFM1 isomer 25.63.
  • Chemically active molecules. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that binds the human transcriptional regulator ZFM1 isomer 25.63.
  • Regular refers to changes in the function of the human transcriptional regulator ZFM1 isoform 25. 63, including increased or decreased protein activity, changes in binding characteristics, and any other organism of the human transcriptional regulator ZFM1 isoform 25. 63 Changes in nature, function, or immunity.
  • Substantially pure ' means essentially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify the human transcriptional regulator ZFM1 isomer 25 using standard protein purification techniques. 63.
  • Substantially pure human transcriptional regulator ZFM1 isomer 25. 63 produces a single main band on a non-reducing polyacrylamide gel.
  • Complementary refers to a polynucleotide that naturally binds by base-pairing under conditions of acceptable salt concentration and temperature.
  • CT-GA 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. The inhibition of such hybridization can be detected by performing hybridization (Southern or Northern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of a completely homologous sequence to a target sequence under conditions of reduced stringency. This does not mean that conditions with reduced stringency allow non-specific binding, because conditions with reduced stringency require that the two sequences interact with each other specifically or selectively.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences based on different methods such as the Clus ter method (Higg ins, DG and PM Sharp (1988) Gene 73: 237-244). 0 The Clus ter method compares each pair by checking the distance between all pairs. Group sequences are arranged in clusters. The clusters are then assigned in pairs or groups.
  • the percent identity between two amino acid sequences is calculated by the following formula: The number of residues matching between sequence A and sequence B
  • the number of spacer residues in B can also be determined by the Clus ter method or by methods known in the art such as Jotun Hein.
  • the percent identity between nucleic acid sequences (Hehi J., (1990) Methods in emzumo logy 183: 625-645).
  • Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitutions for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be a substitution of a hydrogen atom with a fluorenyl, 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, F (ab ') 2 and Fv, which are specific sexually binds to the epitope of the human transcriptional regulator ZFM1 isoform 25.63.
  • 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).
  • 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 in the natural state .
  • isolated human transcriptional regulator ZFM1 isomer 25. 63 refers to the human transcriptional regulator ZFM1 isomer 25. 63 that is substantially free of other proteins, lipids, carbohydrates, or other substance. Those skilled in the art can purify the human transcriptional regulator ZFM1 isomer 25. 63 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. Human transcriptional regulator ZFM1 isomer 25. 63 The purity of the polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, a human transcriptional regulatory factor ZFM1 isomer 25. 63, which basically consists of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques.
  • polypeptide of the invention may be glycosylated, or it 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 the human transcriptional regulator ZFM1 isomer 25.63.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human transcriptional regulatory factor ZFM1 isomer 25.63 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 with another group to include a substituent; or (III) is a type in which a mature polypeptide is fused with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or UV) is a type in which an additional amino acid sequence is fused into a mature polypeptide to form
  • the polypeptide sequences (such as leader sequences or secretory sequences or sequences used to purify this polypeptide or protein 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 full-length polynucleotide sequence of 1989 bases, and its open reading frame of 1178-1879 encodes 233 amino acids. According to the comparison of gene chip expression profiles, it was found that this polypeptide has a similar expression profile with the human transcriptional regulator ZFM1 isomer. It can be inferred that the human transcriptional regulator ZFM1 isomer 25. 63 has the human transcriptional regulator ZFM1 isoform. Functions.
  • the polynucleotide of the present invention may be in the form of DNA or RM.
  • DNA forms include cDNA, genomic MA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • 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.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but having a sequence 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 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.
  • "strict conditions” means: (1) in the lower Hybridization and elution at ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 6 (TC; or (2) hybridization with a denaturant, such as 50% ( ⁇ / ⁇ ) formamide, 0.1% calf serum / 0.1 l »/.
  • 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, 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 the human transcriptional regulator ZFM1 isomer 25.63.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding the human transcriptional regulatory factor ZFM1 isomer 25. 63 of the present invention 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 DM sequence to obtain the double-stranded DNA of the polypeptide.
  • 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 separation of cMA 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. Kits are also commercially available (Qiagene). And the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manua 1, Cold Spooning Harbor Laboratory- New York, 1989). Commercially available cDM libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DM-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of the human transcriptional regulator ZFM1 isomer 25. 63 transcript (4) Detecting the protein product of gene expression by immunological techniques or measuring biological activity. The above methods can be used singly or in combination.
  • 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 nucleosides Acid, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is generally a DM sequence chemically synthesized based on the gene sequence information of the present invention. The genes or fragments of the present invention can of course be used as probes. DM probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product expressing the 25.63 gene of the human transcriptional regulator ZFM1 isomer can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). .
  • a method (Sa ik i, et al. Science 1985; 230: 1350-1354) using DNA technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-c DNA terminal rapid amplification method
  • the primers used for PCR can be based on the polynucleotide sequence information of the present invention disclosed herein. It is appropriately selected and synthesized by a conventional method.
  • 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 determined 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, the sequencing must be repeated. Sometimes it is necessary to determine the cDM sequences of multiple clones in order to splice into full-length cDM sequences.
  • 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 the human transcriptional regulator ZFM1 isomer 25.63 coding sequence, and the present invention is produced by recombinant technology Methods of the polypeptide.
  • a polynucleotide sequence encoding the human transcriptional regulatory factor ZFM1 isomer 25.63 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 (Rosenberg, et al.
  • 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.
  • DM sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis.
  • 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, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenovirus enhancers.
  • 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 the human transcriptional regulatory factor ZFM1 isomer 25.63 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetic engineering containing the polynucleotide or the recombinant vector.
  • Host 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.
  • Escherichia coli, Streptomyces bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells insect cells
  • fly S2 or Sf9 animal cells
  • 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.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing MA can be harvested after the exponential growth phase and treated with CaCl ⁇ .
  • the steps used are well known in the art.
  • MgCl 2 is used.
  • transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DM 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 transcriptional regulator ZFM1 isomer 25. 63 (Science, 1984; 224: 1431). Average There are the following steps:
  • nucleotide or variant
  • encoding the human human transcriptional regulatory factor ZFM1 isomer 25.63 of the present invention or transform or transduce a suitable host cell with a recombinant expression vector containing the polynucleotide ;
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • 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.
  • recombinant proteins can be separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • 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
  • FIG. 1 is a comparison diagram of gene chip expression profiles of the human transcriptional regulator ZFM1 isomer 25. 63 and the human transcriptional regulator ZFM1 isomer according to the present invention.
  • the upper graph is a graph of the expression profile of the human transcriptional regulator ZFM1 isomer 25. 63, and the lower graph is the graph of the expression profile of the human transcriptional regulator ZFM1 isomer.
  • 1-cyste mucosa 2- PMA + Ecv304 cell line, 3- LPS + Ecv304 cell line thymus, 4-normal fibroblasts 1024NC, 5- Fibrob las t, growth factor stimulation, 1024NT, 6- scar into fc Growth factor stimulation, 1013HT, 7-scar into fc without stimulation with growth factor, 1013HC, 8-bladder cancer cell BJ, 9-bladder cancer, 10-bladder cancer, 11-liver cancer, 12-liver cancer cell line, 13 -Fetal skin, 14-spleen, 15-prostate cancer, 16-jejunum adenocarcinoma, 17 cardia cancer.
  • Figure 2 is a polyacrylamide gel electrophoresis image (SDS-PAGE) of the isolated human transcriptional regulator ZFM1 isomer 25.63. 25. 63kDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • CMA was synthesized by reverse transcription reaction using total RM of fetal brain cells as a template and ol igo-dT as a primer. After purification with Qiagene's kit, PCR was performed using the following primers:
  • Pr imer 1 5'- GTGGAAGAGTATTTGGCTTTTCTT-3 '(SEQ ID NO: 3)
  • Pr imer2 5'- CTACAATTTTTTTTATTATGGTAA -3, (SEQ ID NO: 4)
  • Pr imerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Pr imer2 is the 3'-end reverse sequence in SEQ ID NO: 1.
  • This method includes acid guanidinium thiocyanate- Chloroform extraction. That is, the tissue is homogenized with 4M guanidinium isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ), Mix and centrifuge.
  • RNA pellet was washed with 70% ethanol, dried and dissolved in water. Electrophoresis was performed on a 1.2% agarose gel containing 2 g of RNA on 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 mM EDTA-2.2M formaldehyde. Then transferred to nitrocellulose. Preparation of a- 32 P dATP with 32 P- DNA probe labeled by the random primer SYSTEM.
  • Primer 3 5 '-CATGCTAGCATGCCAATACTGGTGGAAAAATTT-3' (Seq ID No: 5)
  • Priraer4 5 '-CCCGAATTCTCACCACCATCCATATCCACAAGT-3' (Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and BamHI restriction sites, respectively , followeded by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively.
  • the restriction sites of Mel and BamHI correspond to the selectivity on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Endonuclease site.
  • the pBS-0396d06 plasmid containing the full-length target gene was used as a template for the PCR reaction.
  • the PCR reaction conditions were as follows: 10 pg of pBS- 0396d06 plasmid, primers ⁇ ! ⁇ 1116: 1: -3 and ⁇ 1 ⁇ 1116: 1: -4 in a total volume of 50 ⁇ 1; 1 ( ⁇ 11101, Advantage polymerase Mix (Clontech) 1 ⁇ 1. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68 ° C 2 min, 25 cycles in total. Ndel and BamHI were used to perform amplification on the product and plasmid pBT-28 (+), respectively.
  • Double-enzyme digestion was used to recover large fragments and ligated with T4 ligase.
  • the ligation product was transformed into Escherichia coli DH5 ⁇ by the calcium chloride method and cultured on LB plates containing kanamycin (final concentration 30 ⁇ ⁇ / ⁇ 1) overnight.
  • the positive clones were screened by colony PCR and sequenced.
  • the positive clones (pET-0396d06) with the correct sequence were selected and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) by calcium chloride method.
  • the host strain BL21 (pET-0396d06) was cultured at 37 ° C in the LB liquid medium (final concentration 30 ⁇ ⁇ / ⁇ 1) at 37 ° C to the logarithmic growth phase, and IPTG was added. Let ol / L reach the final concentration of 1 and continue the cultivation for 5 hours.
  • the bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation, and chromatography was performed using an His. Bind Quick Cartr idge (Novagen) affinity chromatography column capable of binding to 6 histidines (6His-Tag). 63.
  • the purified human transcriptional regulator ZFM1 isomer 25.63 was obtained.
  • Polypeptide synthesizer (product of PE company) was used to synthesize the following human transcriptional regulatory factor ZFM1 isomer 25. 63 specific peptides:
  • NH2-Met-Pro-I le-Leu-Val-Glu-Lys-Phe-Pro-Phe-Val-Arg-Lys-Ser-Glu- COOH SEQ ID NO: 7
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin for methods, see: Avrameas, et al. Immunochemistry, 1969; 6: 43. Rabbits were immunized with 1 ⁇ 2 g of the hemocyanin-peptide complex plus complete Freund's adjuvant, and 15 days later the hemocyanin-polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once.
  • a titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum.
  • Protein A-Sepharose was used to isolate total IgG from antibody-positive rabbit serum.
  • the peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography.
  • the immunoprecipitation method proved that the purified antibody could specifically bind to the human transcriptional regulator ZFM1 isomer 25. 63.
  • Example 6 Application of the polynucleotide fragment of the present invention as a hybridization probe
  • the suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in various aspects.
  • the probes can be used to hybridize to the genome or CDM library 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 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.
  • 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 membrane 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 needle is an oligonucleotide fragment that is completely identical or complementary to the polynucleotide SEQ ID NO: 1 of the present invention;
  • the second type of probe is an oligonucleotide that is partially identical or complementary to the polynucleotide SEQ ID NO: 1 of the present invention Nucleotide fragments.
  • 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.
  • oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes 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 unknown genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used;
  • 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 2 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutant sequence (41Nt) of the gene fragment of SBQ ID NO: 1 or its complementary fragment:
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membrane nitrocellulose membrane
  • Pre-hybridization The sample membrane was placed in a plastic bag, and 3-10 mg of pre-hybridization solution (l OxDenhardt, s; 6xSSC, 0.1 mg / ml CT DM (calf thymus MA)) was added. After the bag was sealed, it was shaken at 68oC with water. 2 hours. cross
  • Gene microarrays or DNA microarrays are new technologies currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as a target DM for gene chip technology for high-throughput research of new gene functions; searching for and screening 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 DeRis i, JL, Lyer, V. & Brown, P. 0. (1997) Science 278, 680-686. And Hel le, RA, Schema, M., Cha i, A., Shalom, 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 polynucleotides of the present invention. Amplify them separately by PCR, and adjust the concentration of the amplified products to At about 500ng / ul, a Cartesian 7500 spotter (purchased from Cartesian Company, USA) was used to spot on the glass medium, and the distance between the spots was 280 ⁇ . The spotted slides were hydrated, dried, and cross-linked in a UV cross-linking instrument. After elution, the DNA was fixed on a glass slide to prepare a chip. The specific method steps have been variously reported in the literature. The post-spot processing steps of this embodiment are:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) by one-step method, and the mRNA was purified with Oligotex mRNA Midi Kit (purchased from QiaGen), and the fluorescent reagents were separately reverse-transcribed.
  • Cy3dUTP (5-Amino-propargyl-2--deoxyuridine 5'-tr iphate cou led to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech) was used to label the mRNA of human mixed tissue
  • Cy5dUTP (5- Amino- propargyl- 2, -deoxyur idine 5'-tr iphate cou led to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech Company, labeled the body's specific tissues (or stimulated cell lines) mRM, and the probes were prepared after purification.
  • the probes from the two types of tissues and the chip were hybridized in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, and a washing solution (1 ⁇ SSC, 0.2% SDS) was used at room temperature. After washing, scanning was performed with a ScanArray 3000 scanner (purchased from General Scanning, USA), and the scanned images were analyzed by Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are bladder mucosa, MA + Ecv304 cell line, LPS + Ecv304 cell line thymus, normal fibroblasts 1024NC, Fibroblast, growth Factor stimulation, 1024NT, scar-to-fc growth factor stimulation, 1013HT, scar-to-fc growth factor stimulation, 1013HC, bladder cancer cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, Prostate cancer, jejunum adenocarcinoma, cardia cancer. Draw a chart based on these 17 Cy3 / Cy5 ratios. (figure 1 ). It can be seen from the figure that the expression profile of the human transcriptional regulator ZFM1 isomer 25.63 and the human transcriptional regulator ZFM1 isomer are very similar. Industrial applicability
  • polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infections and immune diseases.
  • transcriptional regulation of eukaryotic genes is very important for the normal expression of genes, and this regulation process is usually completed by transcriptional regulatory factors.
  • Transcriptional regulators are involved in the body to determine in which tissues and developmental stages genes begin transcription. If a gene encoding such a protein is mutated, not only the gene itself cannot be expressed normally, but also many genes regulated by it cannot be normally transcribed and expressed. There are a large number of different types of transcriptional regulatory factors in higher organisms. These regulatory factors, in combination with various genetic materials DM and RM, regulate the transcription and expression of related genetic materials under the synergistic action of other related proteins. Human transcriptional regulatory factor ZFM1 isoform B3 plays an important role in the transcription and expression of organism genes.
  • the mutation or abnormal expression of this protein will lead to abnormal expression of related proteins in the body and abnormal proliferation of related cells, and then cause various related metabolic and developmental diseases.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of the human transcriptional regulatory factor ZFM1 isomer B3 protein, and both have similar biological functions.
  • the polypeptide of the present invention plays an important regulatory role in the process of transcription and expression of an object gene, and cooperates with some related transcription-activating proteins to regulate the normal transcription and expression of proteins in the body.
  • the mutation or abnormal expression of this protein will lead to abnormal expression of related proteins in the body and abnormal proliferation of related cells, which will lead to the occurrence of disorders of protein metabolism and abnormalities of embryonic development. These diseases include but are not limited to:
  • Disturbances in protein metabolism can affect the following major physiological functions of proteins, leading to the occurrence of related diseases:
  • Protein peptide hormone dysfunction can cause the following diseases: 1) Insulin and glucagon: diabetes, hypoglycemia, etc .;
  • hypothalamus and pituitary hormones Giant disease, dwarfism, acromegaly, Cortisol syndrome (Cushing's syndrome), primary hyperaldosteronism, secondary chronic adrenal insufficiency, hyperthyroidism Hypothyroidism (stingle disease, juvenile hypothyroidism, adult hypothyroidism), male / female infertility, menstrual disorders (functional uterine bleeding, amenorrhea, polycystic ovary syndrome, premenstrual tension syndrome, Menopause syndrome), sexual development disorder, diabetes insipidus, inappropriate antidiuretic hormone secretion syndrome, abnormal lactation, etc .;
  • parathyroid hormone hyperparathyroidism, hypoparathyroidism, etc .
  • Gastrointestinal hormones peptic ulcer, chronic indigestion, chronic gastritis, etc .;
  • Arrhythmia shock, insanity, epilepsy, chorea, hepatic encephalopathy (norepinephrine, Y-aminobutyric acid, serotonin, glutamine), motion sickness, type I allergic disease (net Measles, hay fever, allergic rhinitis, skin allergies), peptic ulcer (histamine), hypercholesterolemia (taurine), tumors (polyamines), etc .;
  • hemoglobin diseases anemia, jaundice, tissue hypoxia-induced organic acidemia
  • various coagulation factor deficiency bleeding
  • muscle spasm muscle forcing
  • muscle paralysis actin
  • Cleft lip most common, with alveolar cleft and cleft palate
  • cleft palate oblique cleft face
  • cervical pouch most common, with alveolar cleft and cleft palate
  • oblique cleft face cervical pouch, cervical palate, etc.
  • Absent in longitudinal direction Absence of upper limb radius / ulnar side, lower limb tibia / fibula side, etc .;
  • Limb differentiation disorder Absence of a certain muscle or muscle group, joint dysplasia, bone deformity, bone fusion, multiple finger (toe) deformity, and finger (toe) deformity, horse tellurium varus etc .;
  • Thyroglossal duct cyst gastrointestinal atresia or stenosis, ileal diverticulum, umbilical diaphragm, congenital umbilical hernia, congenital nonganglioganglion, imperforate anus, abnormal bowel transition, bile duct atresia, circular pancreas, etc
  • neural tube defects no cerebral malformations, spina bifida, spinal meningocele, hydrocephalous meningoencephalocele
  • hydrocephalus inside / outside the brain, etc.
  • the human transcriptional regulatory factor ZFM1 isoform B3 protein plays an extremely important role in the body, which is related to the expression of the MEN1 gene and regulates the differentiation of tumor cells in the body to control the occurrence of malignant diseases such as tumors. Its mutation or abnormal expression is usually closely related to the occurrence of some tumor diseases in the body.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of the human transcriptional regulatory factor ZFM1 isomer B3 protein, and both have similar biological functions.
  • the polypeptide of the present invention regulates the differentiation of tumor cells in a living body in order to control the occurrence of malignant diseases such as tumors, and its mutation or abnormal expression is usually closely related to the occurrence of some tumor diseases in the body. These diseases include but are not limited to:
  • Papilloma squamous cell carcinoma [skin, nasopharynx, larynx, cervix], adenoma (carcinoma) [breast, thyroid], mucinous / serous cystadenomas (carcinoma) [ovarian], basal cell carcinoma [head and face Skin], (malignant) polytype adenoma [extending gland], papilloma, transitional epithelial cancer [bladder, renal pelvis], etc .; 2.
  • Mesenchymal tissue :
  • Malignant lymphoma [Neck, mediastinum, mesenteric and retroperitoneal lymph nodes], various leukemias [lymphoid hematopoietic tissue], multiple myeloma [vertebrae / thorax / ribs / skull and long bones], etc .;
  • Nerve fiber [systemic cutaneous nerve / deep nerve and internal organs], (malignant) schwannoma [nervous of head, neck, limbs, etc.], (malignant) glioblastoma [brain], medulloblastoma [ Cerebellum]
  • malignant melanoma skin, mucous membrane
  • (malignant) hydatidiform mole chorionic epithelial cancer [uterine]
  • (malignant) supporter cells stromal cell tumor
  • (malignant) granulosa cell tumor ovarian, testicular] fine Blastoma [testis], asexual cell tumor [ovary], embryonal cancer [testis, ovary], (malignant) teratoma [ovary, testis, mediastinum and palate tail], etc .
  • malignant melanoma skin, mucous membrane
  • hydatidiform mole chorionic epithelial cancer [uterine]
  • (malignant) supporter cells stromal cell tumor
  • (malignant) granulosa cell tumor ovarian, testicular] fine Blastoma [testis]
  • asexual cell tumor ovary
  • embryonal cancer testis, ovary
  • (malignant) teratoma
  • the invention also provides methods of screening compounds to identify agents that increase (agonist) or repress (antagonist J human transcriptional regulator ZFM1 isomer 25.
  • Agonist enhances human transcriptional regulator ZFM1 isomer 25.
  • 63 Stimulate biological functions such as cell proliferation, and antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • mammalian cells or human expression regulator ZFM1 isomers can be expressed in the presence of drugs 25.
  • the membrane preparation of 63 was cultured with the labeled human transcriptional regulator ZFM1 isomer 25. 63. The ability of the drug to increase or suppress this interaction was then determined.
  • Antagonists of the human transcriptional regulator ZFM1 isomer 25. 63 include antibodies, compounds, receptor deletions, and the like that have been screened.
  • the antagonist of the human transcriptional regulator ZFM1 isomer 25. 63 can bind to the human transcriptional regulator ZFM1 isomer 25. 63 and eliminate its function, or inhibit the production of the polypeptide, or with the active site of the polypeptide Binding prevents the polypeptide from functioning biologically.
  • the human transcriptional regulator ZFM1 isomer 25. 63 can be added to the bioanalytical assay, and by measuring the compound on the human transcriptional regulator ZFM1 isomer 25. 63 and its receptors Effect 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 the human transcriptional regulator ZFM1 isomer 25.63 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, 25.63 molecules of the human transcriptional regulator ZFM1 isomer should generally be labeled. '
  • the present invention provides polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. To produce antibodies. These antibodies can be polyclonal or monoclonal antibodies. The invention also provides antibodies against the human transcriptional regulator ZFM1 isomer 25.63 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • polyclonal antibodies can be obtained by direct injection of the human transcriptional regulatory factor ZFM1 isomer 25.63 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 monoclonal antibodies to the human transcriptional regulator ZFM1 isomer 25. 63 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human B -Cell hybridoma technology, EBV-hybridoma technology, etc.
  • Antibodies against the human transcriptional regulator ZFM1 isomer 25. 63 can be used in immunohistochemical techniques to detect the human transcriptional regulator ZFM1 isomer 25. 63 in biopsy specimens.
  • Monoclonal antibodies that bind to the human transcriptional regulator ZFM1 isomer 25. 63 can also be labeled with radioactive isotopes, 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 the human transcriptional regulator ZFM1 isomer 25 . 63 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to the human transcriptional regulator ZFM1 isomer 25.63.
  • Administration of an appropriate dose of antibody can stimulate or block the production or activity of the human transcriptional regulator ZFM1 isomer 25.63.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting 25.63 levels of the human transcriptional regulatory factor ZFM1 isomer.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human transcription regulator ZFM1 isomer 25. 63 detected in the test can be used to explain the importance of human transcription regulator ZFM1 isomer 25. 63 in various diseases and for the diagnosis of human transcription regulator ZFM1 Diseases where isomers 25.63 work.
  • the 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.
  • the polynucleotide encoding the human transcriptional regulator ZFM1 isomer 25. 63 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 the human transcriptional regulator ZFM1 isomer 25.63.
  • Recombinant gene therapy vectors can be designed to express variant human transcriptional regulator ZFM1 isomer 25. 63, to inhibit the endogenous human transcriptional regulator ZFM1 isoform 25. 63 activity.
  • a variant human transcriptional regulator ZFM1 isomer 25. 63 may be a shortened human transcriptional regulator ZFM1 isomer 25. 63 that lacks a signaling domain, although it can bind to downstream substrates, However, it lacks signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of the human transcriptional regulator ZFM1 isomer 25.63.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding the human transcriptional regulator ZFM1 isomer 25.63 into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding the human transcriptional regulator ZFM1 isomer 25.63 can be found in the literature (Sambrook, etal.).
  • a polynucleotide encoding the human transcriptional regulator ZFM1 isomer 25. 63 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 that inhibit the human transcriptional regulator ZFM1 isomer 25.63 mRNA and ribozymes are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA and performs endonucleation.
  • Antisense RM, DM, and ribozymes can be obtained by any existing RM or DM synthesis technology, such as the technology for the synthesis of oligonucleotides by solid-phase phosphoramidite chemical synthesis, which is widely used.
  • Antisense MA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the MA. This MA 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 phosphorothioate or peptide bond instead of the phosphodiester bond is used for the ribonucleoside linkage.
  • the polynucleotide encoding the human transcriptional regulator ZFM1 isomer 25. 63 can be used for the diagnosis of diseases related to the human transcriptional regulator ZFM1 isomer 25. 63.
  • the polynucleotide encoding the human transcriptional regulator ZFM1 isoform 25. 63 can be used to detect the expression of the human transcriptional regulator ZFM1 isoform 25. 63 or the human transcriptional regulator ZFM1 isoform 25. 63 in a disease state. Abnormal expression.
  • the DNA sequence encoding the human transcription regulatory factor ZFM1 isomer 25. 63 can be used to hybridize biopsy specimens to determine human transcription regulatory factors. ZFMl isomer 25. 63 expression.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and 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 or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • the transcription product of the human transcriptional regulator ZFM1 isomer 25. 63 can also be detected using RNA-polymerase chain reaction (RT-PCR) in vitro amplification with the human transcriptional regulator ZFM1 isomer 25.63 specific primers.
  • Detection of human transcriptional regulator ZFM1 isoform 25 can also be used to diagnose human transcriptional regulator ZFM1 isoform 25. 63-related diseases. Human transcriptional regulator ZFM1 isoform 25. 63 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human transcriptional regulator ZFM1 isoform 25. 63 DNA sequence. Mutations can be detected using existing techniques such as Southern imprinting, DM sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for marking chromosome positions.
  • an important first step is to locate these DNA sequences on a chromosome.
  • a PCR primer (preferably 15-35bp) is prepared from the cDNA, and the sequence can be located on the chromosome. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DM 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 of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • 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, Mendel ian Inher i tance in Man (available through Tohns Hopk ins University Welch Medica Library available online). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cMA sequence-based PCR. 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 transcriptional regulator ZFM1 isomer 25. 63 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human transcriptional regulator ZFM1 isomer 25.63 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.

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Abstract

L'invention concerne un nouveau polypeptide, un isomère humain ZFM1 25.63 du facteur de régulation et de transcription, et un polynucléotide codant 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 de troubles du métabolisme des protéines, de malformations apparaissant lors du développement de l'embryon et de diverses tumeurs communes affectant les tissus. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant le isomère humain ZFM1 25.63 du facteur de régulation et de transcription.
PCT/CN2001/001127 2000-07-07 2001-07-02 Nouveau polypeptide, isomere humain zfm1 25.63 du facteur de regulation et de transcription, et polynucleotide codant ce polypeptide Ceased WO2002020588A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002223372A AU2002223372A1 (en) 2000-07-07 2001-07-02 A novel polypeptide-human transcriptional control factor zfm1 isomer 25.63 and the polynucleotide encoding said polypeptide

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN00117050.3 2000-07-07
CN00117050A CN1333256A (zh) 2000-07-07 2000-07-07 一种新的多肽——人转录调控因子zfm1异构体25.63和编码这种多肽的多核苷酸

Publications (1)

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WO2002020588A1 true WO2002020588A1 (fr) 2002-03-14

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PCT/CN2001/001127 Ceased WO2002020588A1 (fr) 2000-07-07 2001-07-02 Nouveau polypeptide, isomere humain zfm1 25.63 du facteur de regulation et de transcription, et polynucleotide codant ce polypeptide

Country Status (3)

Country Link
CN (1) CN1333256A (fr)
AU (1) AU2002223372A1 (fr)
WO (1) WO2002020588A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07107978A (ja) * 1993-10-13 1995-04-25 Nippon Suisan Kaisha Ltd 魚類プロタミン遺伝子の転写調節因子遺伝子

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07107978A (ja) * 1993-10-13 1995-04-25 Nippon Suisan Kaisha Ltd 魚類プロタミン遺伝子の転写調節因子遺伝子

Also Published As

Publication number Publication date
CN1333256A (zh) 2002-01-30
AU2002223372A1 (en) 2002-03-22

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