WO2002000828A2 - Nouveau polypeptide, proteine a doigt de zinc 34, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, proteine a doigt de zinc 34, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002000828A2
WO2002000828A2 PCT/CN2001/000944 CN0100944W WO0200828A2 WO 2002000828 A2 WO2002000828 A2 WO 2002000828A2 CN 0100944 W CN0100944 W CN 0100944W WO 0200828 A2 WO0200828 A2 WO 0200828A2
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Prior art keywords
polypeptide
polynucleotide
zinc finger
finger protein
sequence
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PCT/CN2001/000944
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English (en)
French (fr)
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WO2002000828A3 (fr
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Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
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Priority to AU89533/01A priority Critical patent/AU8953301A/en
Publication of WO2002000828A2 publication Critical patent/WO2002000828A2/zh
Publication of WO2002000828A3 publication Critical patent/WO2002000828A3/zh

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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

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, a zinc finger protein 34, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a method and application for preparing such polynucleotides and polypeptides. Background technique
  • Transcriptional regulation of eukaryotic genes is very important for the normal expression of genes and exerts biological functions. Usually, transcriptional regulatory factors complete this process. Transcriptional regulatory factors are involved in the body to determine which tissues and developmental stages of genes begin to transcribe. If the genes encoding such proteins are mutated, not only the gene itself cannot be expressed normally, but many genes regulated by it cannot be normal. Perform transcription and expression. The regulation of gene expression by transcription factors is mainly accomplished through the binding of transcription factors to specific DNA sequences, the interaction between transcription factors, and the interaction of transcription factors with conventional transcription mechanisms.
  • DNA binding proteins there are many types of known DNA binding proteins: (1) proteins containing a helix-turn-helix motif; (2) Cys-His zinc finger protein; (3) Cys-Cys zinc finger protein (4) Leu zipper structural protein and so on.
  • Zinc finger protein is a DNA-binding protein, which was first discovered in the amino acid sequence of the protein 5S rRNA gene transcription factor TF III A mediated by Xenopus MA polymerase III. Since then, zinc finger proteins have been expressed in various tissues of different organisms, including hematopoietic cells, brain, nervous system, epidermal tissues, various tissues related to secretion and absorption, and tissues related to tumors and immortal cell lines. Wait. It is estimated that more than 1% of the zinc finger protein genes are in the human genome (Bellefroid et al., 1989; Pellegrino and Berg, 1991).
  • the GATA family is a family of transcription regulators. It can be considered to belong to a zinc finger protein. It is a Cys-Cys zinc finger protein. Zn 2+ forms coordination bonds with four cysteine residues. This domain is required for the GATA transcription factor protein to bind DNA.
  • the binding of GATA transcription factor protein to DM depends on the same sequence on DM: (A / T) GATA (A / G). GATA transcription factor protein can regulate the expression of many genes.
  • GATA-1 GATA-1, GATA-2, GATA-3, GATA- 4 and so on.
  • GATA-1 which can bind to the globin genes expressed in red blood cells and the GATA region of other genes, which plays an important role in the development of red blood cells and the transcriptional regulation of genes in them;
  • GATA-2 regulates the eiidothelin-1 gene in endothelial cells
  • GATA-3 which can bind to the enhancers of alpha and delta genes of T cell receptors, to its regulatory functions;
  • GATA-4 which is expressed in tissues of endothelial origin and cardiac tissue.
  • the human GATA transcription factor protein has important significance for the regulation of the expression of many genes and plays an extremely important role in human life activities.
  • the zinc finger protein 34 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, so there has been a need in the art to identify more zinc involved in these processes.
  • the protein 34 protein especially the amino acid sequence that identifies this protein. Isolation of the new zinc finger protein 34 protein encoding gene 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. Disclosure of invention
  • Another object of the present invention is to provide a method for producing zinc finger protein 34.
  • Another object of the present invention is to provide an antibody against the polypeptide-zinc finger protein 34 of the present invention.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors of the polypeptide-zinc finger protein 34 of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities of zinc finger protein 34.
  • 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 7-924 in SEQ ID NO: 1; and (b) having a position 1430 in SEQ ID NO: 1 the sequence of.
  • the invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • a vector in particular an expression vector, containing the polynucleotide of the invention
  • a host cell genetically engineered with the vector including a transformed, transduced or transfected host cell
  • a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of the zinc finger protein 34 protein, which comprises utilizing the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the present invention also relates to a method for detecting a disease or susceptibility to disease associated with abnormal expression of zinc finger protein 34 protein in vitro, comprising detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, or detecting a mutation in a biological sample.
  • the amount or biological activity of a polypeptide of the invention comprising detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, or detecting a mutation in a biological sample.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the present invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of zinc finger protein 34.
  • Nucleic acid sequence refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and may also refer to genomes or DM or RM, 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 protein or polynucleotide “variant” refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. Variants can have "conservative" changes in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as the replacement of isoleucine with leucine. Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the 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 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 zinc finger protein 34, 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 zinc finger protein 34.
  • Antagonist or an “inhibitor” refers to a molecule that, when combined with zinc finger protein 34, can block or regulate the biological or immunological activity of zinc finger protein 34.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind zinc finger protein 34.
  • Regular refers to a change in the function of zinc finger protein 34, including an increase or decrease in protein activity, a change in binding properties, and any other biological, functional, or immune properties of zinc finger protein 34.
  • substantially pure means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify zinc finger protein 34 using standard protein purification techniques.
  • the substantially pure zinc finger protein 34 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of the zinc finger protein 34 polypeptide can be analyzed by amino acid sequence.
  • “Complementary” or “complementary” refers to a polynucleotide that naturally binds by base-pairing under conditions of acceptable salt concentration and temperature. For example, the sequence "CT-G-A" can be combined with the complementary sequence "G-A-C-T".
  • the complementarity between two single-stranded molecules may be partial or complete. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be achieved by hybridization under conditions of reduced stringency (Sou thern blot or
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences be combined with each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are the same or similar in the comparison of two or more amino acid or nucleic acid sequences.
  • the percent identity can be determined electronically, such as by the MEGAL IGN program (La sergene sof tware package, DNASTAR, Inc., Madi son Wis.).
  • the MEGALIGN program can compare two or more sequences according to different methods such as the Cluster method (Hi gg ins, D. G. and P. M. Sharp (1988) Gene 73: 237-244).
  • the Clus ter method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups.
  • the percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula:
  • the percent identity between nucleic acid sequences can also be determined by the Cluster method or by methods known in the art such as Jotun He in (Hein J., (1990) Methods in emzumo ogy 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.
  • the "antisense strand” refers to a nucleic acid strand that is complementary to the “sense strand”.
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. 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, F (ab ') 2 Fv, which can specifically bind to the epitope of zinc finger protein 34.
  • 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 occurs naturally).
  • a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a vector, or such a polynucleotide or polypeptide may be part of a composition. Since the carrier or composition is not 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 existing in the natural state. .
  • isolated zinc finger protein 34 means that zinc finger protein 34 is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify zinc finger protein 34 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the zinc finger protein 34 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, zinc finger protein 34, 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 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). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of zinc finger protein 34.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the zinc finger protein 34 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 ( ⁇ ) a type in which a group on one or more amino acid residues is replaced 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
  • a polypeptide sequence in which an additional amino acid sequence is fused into the mature polypeptide (Such as leader or secretory sequences or used to purify this poly The sequence of a peptide or protease sequence)
  • such fragments, 00 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 a nucleotide sequence of SBQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence with a total length of 1430 bases, and its open reading frame 7-924 encodes 305 amino acids. According to the comparison of gene chip expression profiles, it was found that this polypeptide has a similar expression profile with zinc finger protein, and it can be concluded that the zinc finger protein 34 has a similar function to zinc finger protein.
  • the polynucleotide of the present invention may be in the form of DM or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DM can be single-stranded or double-stranded.
  • DNA can be a coding or non-coding strand.
  • 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.
  • the "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 that includes the polypeptide and a polynucleotide that includes additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide may 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 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) A denaturant was added during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1 ° /.
  • hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid tablets are 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 nucleotides. Nucleic acid fragments are also Amplification techniques such as PCR can be used to identify and / or isolate a polynucleotide encoding a zinc finger protein 34.
  • 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 zinc finger protein 34 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 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 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 inRNA 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 methods for mRNA extraction, and kits are also commercially available (Qiagene), and constructing cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When combined with polymerase reaction technology, even very 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 DNA-RNA hybrids; (2) the presence or absence of marker gene functions; (3) the level of zinc finger protein 34 transcripts; (4) ) Detection of protein products expressed by genes through immunological techniques or determination of 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 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 2,000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is usually a MA 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 the zinc finger protein 34 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • the RACE method RACE-rapid amplification of cDNA ends
  • the substance can be appropriately selected based on the polynucleotide sequence information of the present invention disclosed herein, and can be 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. In order to obtain the full-length cDNA sequence, sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a zinc finger protein 34 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology.
  • the polynucleotide sequence encoding the zinc finger protein 34 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 for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenoviral enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase, neomycin resistance for eukaryotic cell culture, and And green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase, neomycin resistance for eukaryotic cell culture, and And green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding a zinc finger protein 34 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf 9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DM sequence according to the present invention or a recombinant vector containing the DNA sequence can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and processed by the CaClr method, the steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant zinc finger protein 34 (Sc ience, 1984; 224: 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums according to the host cells used. 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 isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.
  • 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 chromat
  • Fig. 1 is a comparison diagram of gene chip expression profiles of zinc finger protein 34 and zinc finger protein of the present invention.
  • the upper figure is a graph of the zinc finger protein 34 expression profile, and the lower sequence is the zinc finger protein expression profile.
  • 1-bladder mucosa 2-PMA + Ecv304 cell line
  • 3-LPS + Ecv304 cell line thymus 4-normal fibroblasts 1024NC
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated zinc finger protein 34.
  • 34kDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Total RM of human fetal brain was extracted by one step method with guanidine isothiocyanate / phenol / chloroform.
  • Quik mRNA Isolat ion Kit (( ⁇ 6 8 6116 company product) was used to isolate 0 ⁇ (human) mRNA 0 2ug poly (A) mRNA from reverse transcription to form cDNA.
  • Smart cDNA cloning kit (purchased from Clontech) ) The cDNA fragment was inserted into the multicloning site of pBSK (+) vector (Clontech) and transformed into DH5 a to form a cDNA library.
  • CDNA was synthesized using fetal brain total RNA as a template and ol igo-dT as a primer for reverse transcription reaction.
  • Pr imer 1 5'- GACAAAATGTCCCTGGAACAGGAG -3 '(SEQ ID NO: 3)
  • Pr imer2 5'- ATCCTCAACTTGAGACAGGTTTAT -3 '(SEQ ID NO: 4)
  • Pr imerl is a forward sequence starting at lbp at the 5 ′ end of SEQ ID NO: 1;
  • Pr imer2 is the 3'-end reverse sequence in SEQ ID NO: 1.
  • Amplification reaction conditions containing 5 0mmol / L KC 1, 10 ol / L in a reaction volume of 50 ⁇ 1
  • Tr is-CI, (P H8 . 5), 1. 5ramol / L MgCl 2, 200 ⁇ mol / L dNTP, l Opmol primer, 1U Taq DNA polymerase (Clontech Co.).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 55 ° C 30sec; 72 D C 2min.
  • ⁇ -act in was set as a positive control and template blank was set as a negative control.
  • the amplified product was purified using a QIAGEN kit, and ligated to a PCR vector using a TA cloning kit (Invitrogen).
  • the DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as the 1-1430bp shown in SEQ ID NO: 1.
  • Example 3 Nor thern blot analysis of zinc finger protein 34 gene expression:
  • RNA extraction in one step involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue was homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1), centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • RNA Using 20 ⁇ ⁇ RNA, perform electrophoresis on a 1.2% agarose gel containing 20raM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5raM sodium acetate-ImM EDTA-2. 2M formaldehyde . It was then transferred to a nitrocellulose membrane.
  • ⁇ - 32 P dATP was prepared by a random primer method : ' 2 P-labeled DNA probe.
  • the DNA probe used was the PCR amplified zinc finger protein 34 coding region sequence (7bp to 924bp) shown in FIG.
  • the 32P- labeled probe (approximately 2 X 10 6 cpm / ml) and RNA was transferred to a nitrocellulose membrane overnight at 42 ° C in a hybridization solution, the solution comprising 50% formamide - 25mM KH 2 P0 4 (pH7. 4) -5 SSC- 5 ⁇ Denhardt's solution and 200 ⁇ g / 1 ⁇ 2l salmon sperm DNA. After hybridization, the filters were washed in 1 X SSC-0. 1% SDS at 55 C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 4 In vitro expression, isolation and purification of recombinant zinc finger protein 34
  • Primer 3 5'-CCCCATATGATGTCCCTGGAACAGGAGGAGGAA-3 '(Seq ID No: 5)
  • Pr imer4 5'-CATGGATCCTCATGTCACCTGTCCAATCTGTGG-3 '(Seq ID No: 6)
  • the 5' ends of these two primers contain Ndel and BamHI digestion sites, respectively, followed by the coding sequences of the 5 'and 3' ends of the target gene, respectively.
  • Mel and BamHI restriction sites correspond to the expression vector plasmid pET-28b (+) (Novagen Product, Cat. No. 69865. 3).
  • PCR was performed using the pBS-0749c01 plasmid containing the full-length target gene as a template.
  • PCR reaction conditions were as follows: a total volume of 50 ⁇ 1, containing 10 pg of pBS-0749c01 plasmid, 51 3 ⁇ 4 Primer-3 ⁇ PPrimer-4 ⁇ - ⁇ ! 1 Opmol> Advantage polymerase Mix (Clontech) 1 ⁇ 1. 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 coliform bacteria DH5 CC using the calcium chloride method, and cultured overnight in LB plates containing kanamycin (final concentration 3 (Ui g / ral)), and positive clones were selected by colony PCR method and sequenced. Positive sequence correct clone (pET-0749C01) was used to transform the recombinant plasmid into E. coli BL21 (DE3) plySs (product of Novagen) by calcium chloride method.
  • a peptide synthesizer (product of PE company) was used to synthesize the following zinc finger protein 34-specific peptides:
  • 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 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 using a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern blotting, Nor thern blotting, and copying methods. They are all used to fix the polynucleotide sample to be tested on the filter and then hybridize using basically the same steps.
  • 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. 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
  • the GC content is 301 ⁇ 2-70%, and the non-specific hybridization increases when it exceeds;
  • 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 region is compared for homology. If the homology with the non-target molecule region is greater than 85% or there are more than 15 consecutive bases, then the primary probe should not be used;
  • Probe 1 (probel), which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 ( 4 lNt):
  • Probe 2 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutant sequence (41Nt) of the gene fragment of SEQ 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
  • Two NC membranes are required for each probe, so that it can be used in subsequent experiments.
  • the film is washed with high-strength conditions and strength conditions, respectively.
  • probe 1 can be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues.
  • Gene chip or DNA microarray is a new technology that many national laboratories and large pharmaceutical companies are currently developing and developing. 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 fast, efficient, and 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 the literature DeRi s i; J. L., Lyer, Y. & Brown, P. 0.
  • Various cDM total of four full-length nucleotide sequence of more than 000 as a target DNA which comprises a polynucleotide of the present invention. They were amplified by PCR respectively. After purification, the concentration of the amplified product was adjusted to about 500ng / ul, and spotted on a glass medium with a Cartesian 7 500 spotting instrument (purchased from Cartesian Company, USA). The distance between them is 280 ⁇ ⁇ . The spotted slides were hydrated, dried, and cross-linked in a UV cross-linker. After elution, the slides were fixed to fix the DNA on the glass slides to prepare chips.
  • the specific method steps are in the literature There have been many reports in this article.
  • the post-sampling processing steps of this embodiment are:
  • mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and the mRNA was purified using Oligotex mRNA Midi Kit (purchased from QiaGen), and the fluorescence was analyzed by reverse transcription.
  • Cy3dUTP (5-Amino-propargyl-2'-deoxyuridine 5'-triphate coupled to Cy3 fluorescent dye, purchased from Amersham Pharaacia Biotech) was used to label the mRNA of human mixed tissue
  • J Cy5dUTP (5 -Amino-propargyl-2'-deoxyur idine 5'-triphate coupled to Cy5 fluorescent dye (purchased from Amersham Phamacia Biotech) was used to label the specific tissue (or stimulated cell line) tnRNA of the body, and the probe was prepared after purification.
  • the above specific tissues are bladder mucosa, PMA + Ecv304 cell line, LPS + Ecv304 cell line thymus, normal fibroblasts 1G24NC, Fibroblast, growth factor stimulation, 1024NT, scar-like fc growth factor stimulation , 1013HT, scar into fc without stimulation with growth factors, 1013HC, bladder cancer cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunal adenocarcinoma, cardia cancer. Draw a graph based on these Cy3 / Cy5 ratios. (figure 1 ). It can be seen from the figure that the expression profiles of zinc finger protein 34 and zinc finger protein according to the present invention are very similar. Industrial applicability
  • 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.
  • GATA transcription factor protein can regulate the expression of many genes.
  • GATA-1 which can bind to the globin genes expressed in red blood cells and GATA regions of other genes, has an important role in the development of red blood cells and the transcriptional regulation of genes in them; GATA-2, which regulates endothelin in endothelial cells -1 gene expression; GATA-3, which can bind to the enhancer of alpha and delta genes of T cell receptors, to its regulatory function; GATA-4, expressed in tissues of endothelial origin and cardiac tissue. (Trainor CD. Et al., 1990); (Lee ME. Et al., 1991); (Ho IC. Et al., 1991); (Spieth J. et al., 1991).
  • abnormal expression of the specific GATA transcription factor protein raotif will cause abnormal function of the polypeptide containing the raotif of the present invention, resulting in abnormal regulation of the expression of many genes, and related diseases such as red blood cell disease and endothelial tissue disease. , Immune system diseases, tumors, etc.
  • abnormal expression of the zinc finger protein 34 of the present invention will produce various diseases, especially red blood cell disease, endothelial tissue disease, immune system disease, and tumor. These diseases include, but are not limited to:
  • Erythrocyte diseases various anemias such as thalassemia, angiocytic anemia, megaloblastic anemia, malignant anemia, aplastic anemia, hemolytic anemia, myelopathic anemia, anemia secondary to chronic diseases, erythrocytosis, inheritance Oval Red Blood Cells
  • Endothelial Diseases Angiosarcoma, Kaposi's Sarcoma, Hemangioendothelioma, Lymphangioma, Lymphangiomyoma, Heart Valve Disease
  • Immune system diseases Primary T lymphocyte immunodeficiency diseases such as congenital thymic dysplasia, nucleoside phosphorylase deficiency, chronic cutaneous mucosal candidiasis, high IgE syndrome, primary B lymphocyte immunodeficiency disease, Primary cell-humor combined immunodeficiency disease
  • Tumors of various tissues gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioblastoma, Colon cancer, melanoma, adrenal cancer, bladder cancer, bone cancer, osteosarcoma, myeloma, bone marrow cancer, brain cancer, Uterine cancer, endometrial cancer, gallbladder cancer, colon cancer, thymic tumor, nasal cavity and sinus tumor, nasopharyngeal cancer, laryngeal cancer, tracheal tumor, fibroid, fibrosarcoma, lipoma, liposarcoma, leiomyoma
  • Abnormal expression of the zinc finger protein 30 of the present invention will also produce certain inflammations, certain genetic diseases, and the like.
  • the polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially red blood cell diseases, endothelial tissue diseases, immune system diseases, tumors, certain inflammations, certain Some hereditary diseases.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) zinc finger protein 34.
  • Agonists enhance biological functions such as zinc finger protein 34 to stimulate cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing zinc finger protein 34 can be cultured together with labeled zinc finger protein 34 in the presence of a drug. The ability of the drug to increase or block this interaction is then measured.
  • Antagonists of zinc finger protein 34 include antibodies, compounds, receptor deletions, and the like that have been screened.
  • An antagonist of zinc finger protein 34 can bind to zinc finger protein 34 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot perform a biological function.
  • zinc finger protein 34 can be added to a bioanalytical assay to determine whether a compound is an antagonist by measuring the effect of the compound on the interaction between zinc finger protein 34 and its receptor.
  • 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 zinc finger protein 34 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, the zinc finger protein 34 molecule should generally be labeled.
  • the present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies directed against a zinc finger protein 34 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by direct injection of zinc finger protein 34 into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant.
  • Techniques for preparing monoclonal antibodies to zinc finger protein 34 include, but are not limited to, hybridoma technology (Kohl er and
  • Anti-zinc finger protein 34 antibodies can be used in immunohistochemical techniques to detect zinc finger protein 34 in biopsy specimens. Monoclonal antibodies that bind to zinc finger protein 34 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 against a specific bead site in the body.
  • Such as zinc finger protein 34 high affinity monoclonal antibodies can be covalently bound to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of the antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through disulfide exchange. This hybrid antibody can be used to kill zinc finger protein 34-positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to zinc finger protein 34. Administration of an appropriate dose of the antibody can stimulate or block the production or activity of zinc finger protein 34.
  • the present invention also relates to a diagnostic test method for quantitative and localized detection of zinc finger protein 34 levels. These tests are well known in the art and include FISH assays and radioimmunoassays. The level of zinc finger protein 34 detected in the test can be used to explain the importance of zinc finger protein 34 in various diseases and to diagnose diseases where zinc finger protein 34 plays a role.
  • 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.
  • Polynucleotides encoding zinc finger protein 34 can also be used for a variety of therapeutic purposes.
  • Gene therapy technology can be used to treat abnormalities in cell proliferation, development or metabolism caused by the non-expression or abnormal / inactive expression of zinc finger protein 34.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant zinc finger protein 34 to inhibit endogenous zinc finger protein 34 activity.
  • a variant zinc finger protein 34 may be a shortened zinc finger protein 34 that lacks a signaling domain. Although it can bind to downstream substrates, it lacks signaling activity. Therefore, recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of zinc finger protein 34.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus and the like can be used to transfer a polynucleotide encoding a zinc finger protein 34 into a cell.
  • Methods for constructing recombinant viral vectors carrying a polynucleotide encoding a zinc finger protein 34 can be found in the existing literature (Sambrook, et al.).
  • the polynucleotide encoding zinc finger protein 34 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: injecting the polynucleotide directly 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 RNA and DNA
  • ribozymes that inhibit zinc finger protein 34 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target MA to perform endonucleation.
  • Antisense RM, DNA and ribozymes can be obtained by any of the existing RNA or DM synthesis techniques, such as the technology for the synthesis of oligonucleotides by solid-phase phosphoramidite chemical synthesis, which is widely used.
  • Antisense RNA molecules can be expressed in vitro by a DM sequence encoding the RNA Or in vivo transcription. This DNA sequence has been integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of a nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside is linked using a phosphorothioate or peptide bond instead of a phosphodiester bond.
  • a polynucleotide encoding a zinc finger protein 34 can be used in the diagnosis of disease associated with the zinc finger protein 34.
  • the polynucleotide encoding zinc finger protein 34 can be used to detect the expression of zinc finger protein 34 or the abnormal expression of zinc finger protein 34 in a disease state.
  • the DNA sequence encoding zinc finger protein 34 can be used to hybridize biopsy specimens to determine the expression of zinc finger protein 34.
  • Hybridization techniques include Sout hern blotting, No rt hern blotting, in situ hybridization, and the like. These techniques and methods are publicly available and mature, and related kits are commercially available. Part or all of the polynucleotides of the present invention can be immobilized on a microarray as probes
  • Zinc finger protein 34 specific primers can also be used to detect the transcription products of zinc finger protein 34 by RNA-polymerase chain reaction (RT-PCR) in vitro amplification.
  • RT-PCR RNA-polymerase chain reaction
  • Zinc finger protein 34 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild type zinc finger protein 34 DNA sequence. 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 of a human chromosome and can hybridize with it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeat polymorphisms) are available for labeling chromosomal 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 cDNA, and the sequences can be mapped on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells containing human genes corresponding to the primers will produce amplified fragments.
  • 'Somatic cell hybridization is a quick and easy way to map DNA to specific chromosomes.
  • oligonucleotide primers of the present invention by a similar method, 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 Associated with genetic map data. These data can be found, for example, in V. Mckus i ck, Mendel i an Inher i tance in Man (available online with Johns Hopkins Un ivers i ty We l ch Medi ca l L i brary). 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 of the affected individuals and the mutation is not observed in any normal individual, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA 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 that do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which reminders permit their administration on the human body by government agencies that manufacture, use, or sell them.
  • the polypeptide of the present 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.
  • Zinc finger protein 34 is administered in an amount effective to treat and / or prevent a particular indication.
  • the amount and range of zinc finger protein 34 to be 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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Description

一种新的多肽一一锌指蛋白 34和编码这种多肽的多核苷酸 技术领域
本发明属于生物技术领域， 具体地说， 本发明描述了一种新的多肽一一锌指蛋 白 34, 以及编码此多肽的多核苷酸序列。 本发明还涉及此多核苷酸和多肽的制备 方法和应用。 背景技术
真核基因的转录调控对于基因的正常表达及发挥生物学功能是十分重要 的， 通常由转录调控因子来完成这一过程。 转录调控因子在生物体内参与决定基 因在何种组织及何种发育阶段开始转录， 编码这类蛋白的基因如发生突变， 不但 该基因自身不能正常表达， 而且受其调节的许多基因也不能正常的进行转录与表 达。 转录因子对基因表达的调控主要通过转录因子与特定的 DNA序列结合、 转录 因子间的相互作用及转录因子与常规转录机构的相互作用在完成。 根据结构基序 的不同， 已知的 DNA结合蛋白有很多种类： （1 )含有螺旋-转角-螺旋基序的蛋白； ( 2 ) Cys- His锌指蛋白； （3) Cys-Cys锌指蛋白； （4) Leu拉链结构蛋白等等。
锌指蛋白是一种 DNA结合蛋白，最早在非洲爪蟾 MA聚合酶 III介导的 5S rRNA 基因转录因子 TF III A蛋白的氨基酸序列中发现。 此后发现锌指蛋白在不同生物 的各种组织中均有表达， 这些组织包括造血细胞、 脑、 神经系统、 表皮组织、 各 种与分泌吸收相关的组织及与肿瘤和无限增殖细胞系相关的组织等。 据估计在人 类基因组上锌指蛋白基因超过了 1% (Bellefroid et al. , 1989; Pellegrino and Berg, 1991 ) 。
GATA家族是一个转录调控因子家族， 它可以被认为属于一种锌指蛋白， 它是 一种 Cys-Cys锌指蛋白， 其中， Zn2+与四个半胱氨酸残基形成配位键， 这一结构域 对于 GATA转录因子蛋白结合 DNA是必需的。 GATA转录因子蛋白与 DM的结合依 赖于 DM上的一段同样的序列： （A/T)GATA(A/G)， GATA转录因子蛋白可以调控很 多基因的表达。
这个家族的成员包括如下： GATA- 1, GATA- 2, GATA- 3， GATA- 4等等。
几乎所有生物 GATA家族的成员都有一对高度类似的锌指结构，其序列特征是： Cys- x2-Cys-xl7- Cys- x2- Cys。进一步的结构研究发现， GATA家族成员都含有的是 这样一段高度保守的结构域：
C-x- [DN] -C-x (4, 5)— [ST] -χ (2) -W- [HR] - [R ]— χ (3)― [GN]一 χ (3, 4) -C-N- [AS]— C，其 中， 四个 C所代表的半胱氨酸残基是锌指结构的特征配基。 这一结构的高度性证明 其对于 GATA转录因子蛋白发挥正常的生理学功能有着重要的意义。
GATA-1, 它可以结合于红细胞中表达的球蛋白基因以及其他一些基因的 GATA区域， 它对于红细胞的发育和其中基因的转录调控有重要作用； GATA- 2, 调 控内皮细胞中 eiidothelin- 1基因的表达； GATA- 3, 可以结合于 T细胞受体的 alpha 和 delta基因的增强子， 其到调控功能； GATA-4, 在内皮起源的组织和心脏组织 中表达。 （Trainor C. D. , Evans T. , Felsenfeld G. , Boguski M.S. Nature 343: 92-96 (1990) ) ； ( Lee M. E. , Temizer D. T. , Clifford J. A. , Quertermous T. J. Biol. Chem. 266: 16188-16192 (1991) ) ； ( Ho I. - C. , Vorhees P. , Marin N. , Oakley B. K. , Tsai S.— F. , Orkin S. H. ,Liden J. M. EMBO J.
10: 1187-1192 (1991) )； ( Spieth J. , Shim Y. H. , Lea . , Conrad R. , B lumen thai T. Mol. Cell. Biol. 11: 4651-4659 (1991) )
当然， 对人 GATA转录因子蛋白的结构和功能的研究还有待进一步开展， 具体 的内容请参阅相关文献。 （Molecular And Cellular Biology ， Sep 1991 4651-4659 )
综上所述， 人 GATA转录因子蛋白对于很多基因的表达调控有着重要的意义， 在人的生命活动过程中起着极其重要的作用。
通过基因芯片的分析发现，在膀胱粘膜、 PMA+的 Ecv304细胞株、 LPS+的 Ecv304 细胞株胸腺、 正常成纤维细胞 1024NC、 Fibroblast, 生长因子刺激， 1024NT、 疤 痕成 fc生长因子刺激， 1013HT、 疤痕成 fc未用生长因子刺激， 1013HC、 膀胱癌 建株细胞 EJ、 膀胱癌旁、 膀胱癌、 肝癌、 肝癌细胞株、 胎皮、 脾脏、 前列腺癌、 空肠腺癌、 贲门癌中， 本发明的多肽的表达谱与锌指蛋白的表达谱非常近似， 因 此二者功能也可能类似。 本发明被命名为锌指蛋白 34。
由于如上所述锌指蛋白 34蛋白在调节细胞分裂和胚胎发育等机体重要功能中 起重要作用， 而且相信这些调节过程中涉及大量的蛋白， 因而本领域中一直需要 鉴定更多参与这些过程的锌指蛋白 34蛋白， 特别是鉴定这种蛋白的氨基酸序列。 新锌指蛋白 34蛋白编码基因的分离也为研究确定该蛋白在健康和疾病状态下的 作用提供了基础。 这种蛋白可能构成开发疾 1病诊断和 /或治疗药的基础， 因此分 离其编码 DM是非常重要的。 发明的公开
本发明的一个目的是提供分离的新的多肽一一锌指蛋白 34 以及其片段、 类似 物和衍生物。
本发明的另一个目的是提供编码该多肽的多核苷酸。 本发明的另一个目的是提供含有编码锌指蛋白 34的多核苷酸的重组载体。 本发明的另一个目的是提供含有编码锌指蛋白 34的多核苷酸的基因工程化宿 主细胞。
本发明的另一个目的是提供生产锌指蛋白 34的方法。
本发明的另一个目的是提供针对本发明的多肽一一锌指蛋白 34的抗体。
本发明的另一个目的是提供了针对本发明多肽一一锌指蛋白 34的模拟化合 物、 拮抗剂、 激动剂、 抑制剂。
本发明的另一个目的是提供诊断治疗与锌指蛋白 34异常相关的疾病的方法。 本发明涉及一种分离的多肽， 该多肽是人源的， 它包含： 具有 SEQ ID No. 2 氨基酸序列的多肽、 或其保守性变体、 生物活性片段或衍生物。 较佳地， 该多肽 是具有 SEQ ID NO: 2氨基酸序列的多肽。
本发明还涉及一种分离的多核苷酸， 它包含选自下组的一种核苷酸序列或其 变体：
(a)编码具有 SEQ ID No. 2氨基酸序列的多肽的多核苷酸；
(b)与多核苷酸（a)互补的多核苷酸；
(c)与（a)或（b)的多核苷酸序列具有至少 70"/。相同性的多核苷酸。
更佳地，该多核苷酸的序列是选自下组的一种： （a)具有 SEQ I D N0: 1 中 7-924 位的序列； 和（b)具有 SEQ ID NO: 1 中 1 1430位的序列。
本发明另外涉及一种含有本发明多核苷酸的载体， 特别是表达载体； 一种用 该载体遗传工程化的宿主细胞， 包括转化、 转导或转染的宿主细胞； 一种包括培 养所述宿主细胞和回收表达产物的制备本发明多肽的方法。
本发明还涉及一种能与本发明多肽特异性结合的抗体。
本发明还涉及一种筛选的模拟、 激活、 拮抗或抑制锌指蛋白 34蛋白活性的化 合物的方法， 其包括利用本发明的多肽。 本发明还涉及用该方法获得的化合物。
本发明还涉及一种体外检测与锌指蛋白 34 蛋白异常表达相关的疾病或疾病易 感性的方法， 包括检测生物样品中所述多肽或其编码多核苷酸序列中的突变， 或者 检测生物样品中本发明多肽的量或生物活性。
本发明也涉及一种药物组合物， 它含有本发明多肽或其模拟物、 激活剂、 拮抗 剂或抑制剂以及药学上可接受的载体。
本发明还涉及本发明的多肽和 /或多核苷酸在制备用于治疗癌症、发育性疾病 或免疫性疾病或其它由于锌指蛋白 34表达异常所引起疾病的药物的用途。
本发明的其它方面由于本文的技术的公开， 对本领域的技术人员而言是显而易 见的。 本说明书和杈利要求书中使用的下列术语除非特别说明具有如下的含义： "核酸序列" 是指寡核苷酸、 核苷酸或多核苷酸及其片段或部分， 也可以指基 因组或合成的 DM或 RM， 它们可以是单链或双链的， 代表有义链或反义链。 类似 地， 术语 "氨基酸序列" 是指寡肽、 肽、 多肽或蛋白质序列及其片段或部分。 当 本发明中的 "氨基酸序列" 涉及一种天然存在的蛋白质分子的氨基酸序列时， 这 种 "多肽" 或 "蛋白质" 不意味着将氨基酸序列限制为与所述蛋白质分子相关的 完整的天然氨基酸。
蛋白质或多核苷酸 "变体" 是指一种具有一个或多个氨基酸或核苷酸改变的 氨基酸序列或编码它的多核苷酸序列。 所述改变可包括氨基酸序列或核苷酸序列 中氨基酸或核苷酸的缺失、 插入或替换。 变体可具有 "保守性" 改变， 其中替换 的氨基酸具有与原氨基酸相类似的结构或化学性质， 如用亮氨酸替换异亮氨酸。 变体也可具有非保守性改变， 如用色氨酸替换甘氨酸。
"缺失" 是指在氨基酸序列或核苷酸序列中一个或多个氨基酸或核苷酸的缺 失。
"插入" 或 "添加" 是指在氨基酸序列或核苷酸序列中的改变导致与天然存在的 分子相比， 一个或多个氨基酸或核苷酸的增加。 "替换" 是指由不同的氨基酸或核苷 酸替换一个或多个氨基酸或核苷酸。
"生物活性" 是指具有天然分子的结构、 调控或生物化学功能的蛋白质。 类似地, 术语 "免疫学活性" 是指天然的、 重组的或合成蛋白质及其片段在合适的动物或细胞 中诱导特定免疫反应以及与特异性抗体结合的能力。
"激动剂"是指当与锌指蛋白 34结合时，一种可引起该蛋白质改变从而调节该 蛋白质活性的分子。 激动剂可以包括蛋白质、 核酸、 碳水化合物或任何其它可结 合锌指蛋白 34的分子。
"拮抗剂" 或' "抑制物"是指当与锌指蛋白 34结合时， 一种可封闭或调节锌指 蛋白 34的生物学活性或免疫学活性的分子。 拮抗剂和抑制物可以包括蛋白质、 核 酸、 碳水化合物或任何其它可结合锌指蛋白 34的分子。
"调节"是指锌指蛋白 34的功能发生改变， 包括蛋白质活性的升高或降低、 结 合特性的改变及锌指蛋白 34的任何其它生物学性质、 功能或免疫性质的改变。
"基本上纯"是指基本上不含天然与其相关的其它蛋白、 脂类、 糖类或其它物质。 本领域的技术人员能用标准的蛋白质纯化技术纯化锌指蛋白 34。 基本上纯的锌指蛋 白 34在非还原性聚丙烯酰胺凝胶上能产生单一的主带。锌指蛋白 34多肽的纯度可用 氨基酸序列分析。 "互补的" 或 "互补"是指在允许的盐浓度和温度条件下通过碱基配对的多核 苷酸天然结合。 例如， 序列 "C-T- G- A" 可与互补的序列 "G- A- C- T" 结合。 两个 单链分子之间的互补可以是部分的或全部的。 核酸链之间的互补程度对于核酸链 之间杂交的效率及强度有明显影响。
"同源性"是指互补的程度， 可以是部分同源或完全同源。 "部分同源"是指 一种部分互补的序列， 其至少可部分抑制完全互补的序列与靶核酸的杂交。 这种 杂交的抑制可通过在严格性程度降低的条件下进行杂交 （Sou thern印迹或
Nor thern印迹等） 来检测。 基本上同源的序列或杂交探针可竟争和抑制完全同源 的序列与靶序列在的严格性程度降低的条件下的结合。 这并不意味严格性程度降 低的条件允许非特异性结合， 因为严格性程度降低的条件要求两条序列相互的结 合为特异性或选择性相互作用。
"相同性百分率"是指在两种或多种氨基酸或核酸序列比较中序列相同或相似 的百分率。 可用电子方法测定相同性百分率， 如通过 MEGAL IGN程序 （La sergene sof tware package, DNASTAR, Inc. , Madi son Wi s. ) 。 MEGALIGN程序可根据不同 的方法如 C lus ter法比较两种或多种序列（Hi gg ins , D. G. 和 P. M. Sharp (1988) Gene 73: 237-244)。 Clus ter法通过检查所有配对之间的距离将各组序列排列成 簇。 然后将各簇以成对或成组分配。 两个氨基酸序列如序列 A和序列 B之间的相同 性百分率通过下式计算：
序列 A与序列 B之间匹配的残基个数 X 100 序列 A的残基数一序列 A中间隔残基数一序列 B中间隔残基数
也可以通过 C lus t er法或用本领域周知的方法如 Jotun He in 测定核酸序列之 间的相同性百分率（Hein J. , (1990) Methods in emzumo l ogy 183: 625—645)。
"相似性" 是指氨基酸序列之间排列对比时相应位置氨基酸残基的相同或保 守性取代的程度。 用于保守性取代的氨基酸例如， 带负电荷的氨基酸可包括天冬 氨酸和谷氨酸； 带正电荷的氨基酸可包括赖氨酸和精氨酸； 具有不带电荷的头部 基团有相似亲水性的氨基酸可包括亮氨酸、 异亮氨酸和缬氨酸； 甘氨酸和丙氨酸; 天冬酰胺和谷氨酰胺； 丝氨酸和苏氨酸； 苯丙氨酸和酪氨酸。
"反义"是指与特定的 DNA或 RNA序列互补的核苷酸序列。 "反义链"是指与 "有 义链" 互补的核酸链。
"衍生物" 是指 HFP或编码其的核酸的化学修饰物。 这种化学修饰物可以是用 烷基、 酰基或氨基替换氢原子。 核酸衍生物可编码保留天然分子的主要生物学特 性的多肽。 "抗体" 是指完整的抗体分子及其片段， 如 Fa、 F (ab') 2 Fv , 其能特异性结 合锌指蛋白 34的抗原决定簇。
"人源化抗体" 是指非抗原结合区域的氨基酸序列被替换变得与人抗体更为 相似， 但仍保留原始结合活性的抗体。 .
"分离的"一词指将物质从它原来的环境（例如， 若是自然产生的就指其天然 环境） 之中移出。 比如说， 一个自然产生的多核苷酸或多肽存在于活动物中就是 没有被分离出来， 但同样的多核苷酸或多肽同一些或全部在自然系统中与之共存 的物质分开就是分离的。 这样的多核苷酸可能是某一载体的一部分， 也可能这样 的多核苷酸或多肽是某一组合物的一部分。 既然载体或组合物不是它天然环境的 成分， 它们仍然是分离的。
如本发明所用， "分离的" 是指物质从其原始环境中分离出来 （如果是天然 的物质， 原始环境即是天然环境） 。 如活体细胞内的天然状态下的多聚核苷酸和 多肽是没有分离纯化的， 但同样的多聚核苷酸或多肽如从天然状态中同存在的其 他物质中分开， 则为分离纯化的。
如本文所用， "分离的锌指蛋白 34"是指锌指蛋白 34基本上不含天然与其相 关的其它蛋白、 脂类、 糖类或其它物质。 本领域的技术人员能用标准的蛋白质纯 化技术纯化锌指蛋白 34。 基本上纯的多肽在非还原聚丙烯酰胺凝胶上能产生单一 的主带。 锌指蛋白 34多肽的纯度能用氨基酸序列分析。
本发明提供了一种新的多肽一一锌指蛋白 34 , 其基本上是由 SEQ ID N0: 2所示的 氨基酸序列组成的。 本发明的多肽可以是重组多肽、 天然多肽、 合成多肽， 优选重 组多肽。 本发明的多肽可以是天然纯化的产物， 或是化学合成的产物， 或使用重组 技术从原核或真核宿主 (例如， 细菌、 酵母、 高等植物、 昆虫和哺乳动物细胞）中产 生。 根据重组生产方案所用的宿主， 本发明的多肽可以是糖基化的， 或可以是非糖 基化的。 本发明的多肽还可包括或不包括起始的甲硫氨酸残基。
本发明还包括锌指蛋白 34的片段、衍生物和类似物。如本发明所用，术语"片 段" 、 "衍生物" 和 "类似物" 是指基本上保持本发明的锌指蛋白 34相同的生物 学功能或活性的多肽。 本发明多肽的片段、 衍生物或类似物可以是： （I )这样一 种， 其中一个或多个氨基酸残基被保守或非保守氨基酸残基 （优选的是保守氨基 酸残基） 取代， 并且取代的氨基酸可以是也可以不是由遗传密码子编码的； 或者 ( Π ) 这样一种， 其中一个或多个氨基酸残基上的某个基团被其它基团取代包含 取代基； 或者（ Π Ι )这样一种， 其中成熟多肽与另一种化合物 （比如延长多肽半 衰期的化合物， 例如聚乙二醇） 融合； 或者 （ IV ) 这样一种， 其中附加的氨基酸 序列融合进成熟多肽而形成的多肽序列 （如前导序列或分泌序列或用来纯化此多 肽的序列或蛋白原序列）通过本文的阐述， 这样的片段、 00衍生物和类似物被认 为在本领域技术人员的知识范围之内。
本发明提供了分离的核酸 （多核苷酸） ， 基本由编码具有 SEQ ID NO: 2 氨基 酸序列的多肽的多核苷酸组成。 本发明的多核苷酸序列包括 SBQ ID NO: 1的核苷 酸序列。 本发明的多核苷酸是从人胎脑组织的 cDNA文库中发现的。 它包含的多核 苷酸序列全长为 1430个碱基， 其开放读框 7- 924编码了 305个氨基酸。 根据基因 芯片表达谱比较发现， 此多肽与锌指蛋白有相似的表达谱， 可推断出该锌指蛋白 34具有锌指蛋白相似的功能。
本发明的多核苷酸可以是 DM形式或是 RNA形式。 DNA形式包括 cDNA、基因组 DNA或人工合成的 DNA。 DM可以是单链的或是双链的。 DNA可以是编码链或非编 码链。 编码成熟多肽的编码区序列可以与 SEQ ID NO: 1所示的编码区序列相同或 者是简并的变异体。 如本发明所用， "简并的变异体" 在本发明中是指编码具有 SEQ ID NO: 2的蛋白质或多肽， 但与 SEQ ID NO: 1所示的编码区序列有差别的核 酸序列。
编码 SEQ ID N0: 2的成熟多肽的多核苷酸包括： 只有成熟多肽的编码序列； 成熟多肽的编码序列和各种附加编码序列； 成熟多肽的编码序列 （和任选的附加 编码序列） 以及非编码序列。
术语 "编码多肽的多核苷酸" 是指包括编码此多肽的多核苷酸和包括附加编 码和 /或非编码序列的多核苷酸。
本发明还涉及上述描述多核苷酸的变异体， 其编码与本发明有相同的氨基酸 序列的多肽或多肽的片断、 类似物和衍生物。 此多核苷酸的变异体可以是天然发 生的等位变异体或非天然发生的.变异体。 这些核苷酸变异体包括取代变异体、 缺 失变异体和插入变异体。 如本领域所知的， 等位变异体是一个多核苷酸的替换形 式， 它可能是一个或多个核苷酸的取代、 缺失或插入， 但不会从实质上改变其编 码的多肽的功能。
本发明还涉及与以上所描述的序列杂交的多核苷酸 （两个序列之间具有至少 50% , 优选具有 70%的相同性） 。 本发明特别涉及在严格条件下与本发明所述多核 苷酸可杂交的多核苷酸。 在本发明中， "严格条件" 是指： （1)在较低离子强度和 较高温度下的杂交和洗脱， 如 0. 2xSSC, 0. 1%SDS, 60 °C ;或（2)杂交时加用变性剂， 如 50% (v/v)甲酰胺， 0. 1%小牛血清 / 0. l°/。F i co l l , 42 °C等； 或（3)仅在两条序列之 间的相同性至少在 95%以上，更好是 97½以上时才发生杂交。 并且， 可杂交的多核 苷酸编码的多肽与 SEQ ID NO: 2所示的成熟多肽有相同的生物学功能和活性。
本发明还涉及与以上所描述的序列杂交的核酸片段。 如本发明所用， "核酸片 段"的长度至少含 10个核苷酸， 较好是至少 20- 30个核苷酸， 更好是至少 50-60 个核苷酸， 最好是至少 100个核苷酸以上。 核酸片段也可用于核酸的扩增技术（如 PCR)以确定和 /或分离编码锌指蛋白 34的多核苷酸。
本发明中的多肽和多核苷酸优选以分离的形式提供， 更佳地被纯化至均质。 本发明的编码锌指蛋白 34的特异的多核苷酸序列能用多种方法获得。 例如， 用本领域熟知的杂交技术分离多核苷酸。 这些技术包括但不局限于： 1)用探针与 基因组或 cDNA文库杂交以检出同源的多核苷酸序列，和 2)表达文库的抗体筛选以 检出具有共同结构特征的克隆的多核苷酸片段。
本发明的 DNA片段序列也能用下列方法获得： 1)从基因组 DNA分离双链 DNA 序列； 2)化学合成 DNA序列以获得所述多肽的双链 DNA。
上述提到的方法中， 分离基因组 DM最不常用。 DNA序列的直接化学合成是经 常选用的方法。 更经常选用的方法是 cDNA序列的分离。 分离感兴趣的 cDNA的标 准方法是从高表达该基因的供体细胞分离 inRNA并进行逆转录， 形成质粒或噬菌体 cDNA文库。 提取 mRNA的方法已有多种成熟的技术， 试剂盒也可从商业途径获得 (Qiagene) , 而构建 cDNA文库也是通常的方法（Sambrook, et al. , Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989)。 还可得到商业供应的 cDNA文库， 如 Clontech公司的不同 cDNA文库。 当 结合使用聚合酶反应技术时， 即使极少的表达产物也能克隆。
可用常规方法从这些 cDNA文库中筛选本发明的基因。 这些方法包括（但不限 于）： （l)DNA-DNA或 DNA-RNA杂,交； （2)标志基因功能的出现或丧失； （3)测定锌指 蛋白 34的转录本的水平； （4)通过免疫学技术或测定生物学活性， 来检测基因表 达的蛋白产物。 上述方法可单用， 也可多种方法联合应用。
在第（1)种方法中， 杂交所用的探针是与本发明的多核苷酸的任何一部分同 源，其长度至少 10个核苷酸， 较好是至少 30个核苷酸， 更好是至少 50个核苷酸， 最好是至少 100个核苷酸。 此外， 探针的长度通常在 2000个核苷酸之内， 较佳的 为 1000个核苷酸之内。 此处所用的探针通常是在本发明的基因序列信息的基础上 化学合成的 MA序列。 本发明的基因本身或者片段当然可以用作探针。 DNA探针的 标记可用放射性同位素， 荧光素或酶（如碱性磷酸酶）等。
在第（4)种方法中， 检测锌指蛋白 34基因表达的蛋白产物可用免疫学技术如 Western印迹法， 放射免疫沉淀法， 酶联免疫吸附法（ELISA)等。
应用 PCR技术扩增 DM/RNA的方法（Saiki, et al. Science
1985; 230: 1350- 1354)被优选用于获得本发明的基因。 特别是很难从文库中得到全 长的 cDNA时， 可优选使用 RACE法（RACE- cDNA末端快速扩增法）， 用于 PCR的引 物可根据本文所公开的本发明的多核苷酸序列信息适当地选择， 并可用常规方法 合成。 可用常规方法如通过凝胶电泳分离和纯化扩增的 DNA/RNA片段。
如上所述得到的本发明的基因， 或者各种 DNA片段等的多核苷酸序列可用常 规方法如双脱氧链终止法（Sanger et al. PNAS, 1977, 74： 5463- 5467)测定。 这 类多核苷酸序列测定也可用商业测序试剂盒等。 为了获得全长的 cDNA序列， 测序 需反复进行。 有时需要测定多个克隆的 cDNA序列， 才能拼接成全长的 cDNA序列。
本发明也涉及包含本发明的多核苷酸的载体， 以及用本发明的载体或直接用 锌指蛋白 34编码序列经基因工程产生的宿主细胞， 以及经重组技术产生本发明所 述多肽的方法。
本发明中， 编码锌指蛋白 34的多核苷酸序列可插入到载体中， 以构成含有本 发明所述多核苷酸的重组载体。 术语 "载体" 指本领域熟知的细菌质粒、 噬菌体、 酵母质粒、 植物细胞病毒、 哺乳动物细胞病毒如腺病毒、 逆转录病毒或其它载体。 在本发明中适用的载体包括但不限于： 在细菌中表达的基于 T7启动子的表达载体 (Rosenberg, et al. Gene, 1987, 56: 125)； 在哺乳动物细胞中表达的 pMSXND表 达载体（Lee and Nathans, J Bio Chem. 263: 3521， 1988)和在昆虫细胞中表达的 来源于杆状病毒的载体。 总之， 只要能在宿主体内复制和稳定， 任何质粒和载体 都可以用于构建重组表达载体。 表达载体的一个重要特征是通常含有复制起始点、 启动子、 标记基因和翻译调控元件。
本领域的技术人员熟知的方法能用于构建含编码锌指蛋白 34的 DNA序列和合 适的转录 /翻译调控元件的表达载体。 这些方法包括体外重组 DNA技术、 DNA合成 技术、 体内重组技术等（Sanibroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989)。 所述的 DM序列 可有效连接到表达载体中的适当启动子上， 以指导 mRNA合成。 这些启动子的代表 性例子有： 大肠杆菌的 lac或 trp启动子； λ噬菌体的 PL启动子； 真核启动子包 括 CMV立即早期启动子、 HSV胸苷激酶启动子、 早期和晚期 SV40启动子、 反转录 病毒的 LTRs和其它一些已知的可控制基因在原核细胞或真核细胞或其病毒中表达 的启动子。 表达载体还包括翻译起始用的核糖体结合位点和转录终止子等。 在载 体中插入增强子序列将会使其在高等真核细胞中的转录得到增强。 增强子是 DNA 表达的顺式作用因子， 通常大约有 10到 300个碱基对， 作用于启动子以增强基因 的转录。 可举的例子包括在复制起始点晚期一侧的 100到 270个碱基对的 SV40增 强子、 在复制起始点晚期一侧的多瘤增强子以及腺病毒增强子等。
此外， 表达载体优选地包含一个或多个选择性标记基因， 以提供用于选择转 化的宿主细胞的表型性状， 如真核细胞培养用的二氢叶酸还原酶、 新霉素抗性以 及绿色荧光蛋白（GFP) , 或用于大肠杆菌的四环素或氨苄青霉素抗性等。
本领域一般技术人员都清楚如何选择适当的载体 /转录调控元件（如启动子、 增强子等） 和选择性标记基因。
本发明中， 编码锌指蛋白 34的多核苷酸或含有该多核苷酸的重组载体可转化 或转导入宿主细胞， 以构成含有该多核苷酸或重组载体的基因工程化宿主细胞。 术语 "宿主细胞" 指原核细胞， 如细菌细胞； 或是低等真核细胞， 如酵母细胞； 或是高等真核细胞， 如哺乳动物细胞。 代表性例子有： 大肠杆菌， 链霉菌属； 细 菌细胞如鼠伤寒沙门氏菌；真菌细胞如酵母；植物细胞；昆虫细胞如果蝇 S2或 Sf 9; 动物细胞如 CH0、 COS或 Bowes黑素瘤细胞等。
用本发明所述的 DM序列或含有所述 DNA序列的重组载体转化宿主细胞可用 本领域技术人员熟知的常规技术进行。 当宿主为原核生物如大肠杆菌时， 能吸收 DNA的感受态细胞可在指数生长期后收获， 用 CaClr法处理， 所用的步骤在本领域 众所周知。 可供选择的是用 MgCl2。 如果需要， 转化也可用电穿孔的方法进行。 当 宿主是真核生物， 可选用如下的 DNA转染方法： 磷酸钙共沉淀法， 或者常规机械 方法如显微注射、 电穿孔、 脂质体包装等。
通过常规的重组 DNA技术， 利用本发明的多核苷酸序列可用来表达或生产重 组的锌指蛋白 34 (Sc ience, 1984; 224： 1431 )。 一般来说有以下步骤：
(1) .用本发明的编码人 锌指蛋白 34的多核苷酸 (或变异体）， 或用含有该多 核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2) .在合适的培养基中培养宿主细胞；
(3) .从培养基或细胞中分离、 纯化蛋白质。
在步骤 （2 ) 中， 根据所用的宿主细胞， 培养中所用的培养基可选自各种常 规培养基。 在适于宿主细胞生长的条件下进行培养。 当宿主细胞生长到适当的细 胞密度后， 用合适的方法（如温度转换或化学诱导）诱导选择的启动子， 将细胞再 培养一段时间。
在步骤（ 3 ) 中， 重组多肽可包被于细胞内、 或在细胞膜上表达、 或分泌到细 胞外。 如果需要， 可利用其物理的、 化学的和其它特性通过各种分离方法分离和 纯化重组的蛋白。 这些方法是本领域技术人员所熟知的。 这些方法包括但并不限 于： 常规的复性处理、 蛋白沉淀剂处理（盐析方法）、 离心、 渗透破菌、 超声波处 理、 超离心、 分子筛层析（凝胶过滤）、 吸附层析、 离子交换层析、 高效液相层析 (HPLC)和其它各种液相层析技术及这些方法的结合。 附图的简要说明 下列附图用于说明本发明的具体实施方案， 而不用于限定由杈利要求书所界 定的本发明范围。
图 1是本发明锌指蛋白 34和锌指蛋白的基因芯片表达谱比较图。上图是锌指蛋白 34的表达谱折方图，下方序列是锌指蛋白的表达谱折方图。其中， 1 -膀胱粘膜、 2-PMA+ 的 Ecv304细胞株、 3- LPS+的 Ecv304细胞株胸腺、 4-正常成纤维细胞 1024NC、
5-Fibroblas t , 生长因子刺激， 1024NT, 6-疤痕成 fc生长因子刺激， 1013HT, 7-疤 痕成 fc未用生长因子刺激， 1013HC、 8-膀胱癌建株细胞 EJ、 9-膀胱癌旁、 10-膀胱癌、 11 -肝癌、 12-肝癌细胞株、 13-胎皮、 14-脾脏、 15-前列腺癌、 16-空肠腺癌、 17贲 门癌。
图 2为分离的锌指蛋白 34的聚丙烯酰胺凝胶电泳图 （SDS- PAGE ) 。 34kDa为蛋白 质的分子量。 箭头所指为分离出的蛋白条带。 实现本发明的最佳
下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明本 发明而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通 常按照常规条件如 Sambrook等人， 分子克隆：实验室手册（New York: Co l d Spr ing Harbor Labora t ory Pres s , 1989)中所述的条件， 或按照制造厂商所建议的条件。 实施例 1 : 锌指蛋白 34的克隆
用异硫氰酸胍 /酚 /氯仿一步法提取人胎脑总 RM。 用 Quik mRNA Isolat ion Ki t ( (^ 686116 公司产品）从总1 中分离 0 ^ (人） mRNA0 2ug poly (A) mRNA经逆转录形 成 cDNA。 用 Smart cDNA克隆试剂盒 (购自 Clontech ) 将 cDNA片段定向插入到 pBSK (+) 载体（C lontech公司产品）的多克隆位点上， 转化 DH5 a , 细菌形成 cDNA文库。 用 Dye termina te cycle react ion sequencing ki t (Perkin-Elmer公司产品) 和 ABI 377自 动测序仪 (Perkin- Elmer公司）测定所有克隆的 5'和 3'末端的序列。将测定的 cDNA序列 与巳有的公共 DNA序列数据库（Genebank )进行比较， 结果发现其中一个克隆 0749c01 的 cDNA序列为新的 DNA。 通过合成一系列引物对该克隆所含的插入 cDNA片段进行双向 测定。 结果表明， 0749c01克隆所含的全长 cDNA为 1430bp (如 Seq ID NO: 1所示） ， 从 第 7bp至 924bp有一个 918bp的开放阅读框架 ( 0RF ) ' 编码一个新的蛋白质（如 Seq ID N0: 2所示） 。 我们将此克隆命名为 pBS-0749c01 , 编码的蛋白质命名为锌指蛋白 34。 实施例 2: 用 RT-PCR方法克隆编码锌指蛋白 34的基因
用胎脑细胞总 RNA为模板，以 ol igo-dT为引物进行逆转录反应合成 cDNA,用
Qiagene的试剂盒纯化后，用下列引物进行 PCR扩增： Pr imer 1: 5'- GACAAAATGTCCCTGGAACAGGAG -3' (SEQ ID NO: 3)
Pr imer2: 5'- ATCCTCAACTTGAGACAGGTTTAT -3' (SEQ ID NO: 4)
Pr imerl为位于 SEQ ID NO: 1的 5'端的第 lbp开始的正向序列；
Pr imer2为 SEQ ID NO: 1的中的 3'端反向序列。
扩增反应的条件： 在 50 μ 1的反应体积中含有 50mmol/L KC 1, 10 o l/L
Tr is-CI, (PH8. 5) , 1. 5ramol/L MgCl2, 200 μ mol/L dNTP, l Opmol引物， 1U的 Taq DNA聚 合酶（Clontech公司产品）。 在 PE9600型 DNA热循环仪（Perkin- Elmer公司）上按下列条 件反应 25个周期： 94°C 30sec; 55°C 30sec; 72DC 2min。 在 RT-PCR时同时设 β -act in 为阳性对照和模板空白为阴性对照。 扩增产物用 QIAGEN公司的试剂盒纯化， 用 TA克隆 试剂盒连接到 PCR载体上（Invi trogen公司产品） 。 DNA序列分析结果表明 PCR产物的 DNA序列与 SEQ ID NO: 1所示的 1- 1430bp完全相同。 实施例 3: Nor thern 印迹法分析锌指蛋白 34基因的表达：
用一步法提取总 RNA [Anal. Biochem 1987, 162, 156-159]。 该法包括酸性硫氰酸 胍苯酚 -氯仿抽提。 即用 4M异硫氰酸胍- 25mM柠檬酸钠， 0. 2M乙酸钠 （ pH4. 0 ) 对组织 进行匀浆， 加入 1倍体积的苯酚和 1/5体积的氯仿-异戊醇（49 : 1 ) , 混合后离心。 吸 出水相层， 加入异丙醇 （0. 8体积） 并将混合物离心得到 RNA沉淀。 将得到的 RNA沉淀 用 70%乙醇洗涤， 干燥并溶于水中。 用 20 μ § RNA, 在含 20raM 3- ( N-吗啉代）丙磺酸 ( pH7. 0 ) - 5raM乙酸钠 - ImM EDTA-2. 2M甲醛的 1. 2%琼脂糖凝胶上进行电泳。 然后转移 至硝酸纤维素膜上。用 α -32P dATP通过随机引物法制备:'2P-标记的DNA探针。所用的 DNA 探针为图 1所示的 PCR扩增的锌指蛋白 34编码区序列（7bp至 924bp)。将 32P-标记的探针 (约 2 X 106cpm/ml )与转移了 RNA的硝酸纤维素膜在一溶液中于 42°C杂交过夜， 该溶液 包含 50%甲酰胺- 25mM KH2P04 ( pH7. 4 ) -5 SSC- 5 χ Denhardt 's溶液和 200 μ g/½l鲑精 DNA。杂交之后，将滤膜在 1 X SSC-0. 1%SDS中于 55 C洗 30min。然后,用 Phosphor Imager 进行分析和定量。 实施例 4: 重组锌指蛋白 34的体外表达、 分离和纯化
根据 SEQ ID NO: 1和图 1所示的编码区序列， 设计出一对特异性扩增引物， 序列如 下：
Primer 3: 5'-CCCCATATGATGTCCCTGGAACAGGAGGAGGAA-3' ( Seq ID No: 5 )
Pr imer4: 5'-CATGGATCCTCATGTCACCTGTCCAATCTGTGG-3' ( Seq ID No: 6 ) 此两段引物的 5'端分别含有 Ndel和 BamHI酶切位点，其后分别为目的基因 5'端和 3' 端的编码序列， Mel和 BamHI酶切位点相应于表达载体质粒 pET- 28b (+) (Novagen公司 产品， Cat. No. 69865. 3)上的选择性内切酶位点。 以含有全长目的基因的 pBS-0749c01 质粒为模板，进行 PCR反应。 PCR反应条件为： 总体积 50 μ 1中含 pBS- 0749c01质粒 10pg、 51 ¾Pr imer-3^PPrimer-4^-^!) 1 Opmol > Advantage polymerase Mix ( Cl ontech公司 产品） 1 μ 1。 循环参数： 94°C 20s, 60°C 30s, 68°C 2 min，共 25个循环。 用 Ndel和 BamHI 分别对扩增产物和质粒 PET- 28 (+)进行双酶切，分别回收大片段,并用 T4连接酶连接。 连接产物转化用氯化钙法大肠杆细菌 DH5 CC ,在含卡那霉素 （终浓度 3 (Ui g/ral ) 的 LB 平板培养过夜后， 用菌落 PCR方法筛选阳性克隆， 并进行测序。 挑选序列正确的阳性 克隆（pET- 0749C01 )用氯化钙法将重组质粒转化大肠杆菌 BL21 (DE3) plySs (Novagen 公司产品）。 在含卡那霉素 （终浓度 30 y g/ml ) 的 LB液体培养基中， 宿主菌 BL21 ( PET- 0749C01 ) 在 37°C培养至对数生长期， 加入 IPTG至终浓度 lmmol/L， 继续培养 5 小时。 离心收集菌体， 经超声波破菌，离心收集上清， 用能与 6个组氨酸（6Hi s- Tag ) 结合的亲和层析柱 Hi s. Bind Quick Cartridge ( Novagen公司产品） 进行层析， 得到 了纯化的目的蛋白锌指蛋白 34。经 SDS-PAGE电泳，在 34kDa处得到一单一的条带（图 2 )。 将该条带转移至 PVDF膜上用 Edams水解法进行 N-端氨基酸序列分析， 结果 N-端 15个氨 基酸与 SEQ ID NO: 2所示的 N-端 15个氨基酸残基完全相同。 实施例 5 抗锌指蛋白 34抗体的产生
用多肽合成仪 （PE公司产品）合成下述锌指蛋白 34特异性的多肽：
NH2-Met-S e r-Leu-G 1 u-G 1 n- G 1 u-G 1 u-G 1 u-Thr-G 1 n- P r o-G 1 y-Arg-Leu-Leu-C00H (SEQ ID N0: 7)。 将该多肽分别与血蓝蛋白和牛血清白蛋白耦合形成复合， 方法参见： Avrameas, et a l . Ira麵 ochemi s try, 1969; 6: 43。 用 4mg上述血蓝蛋白多肽复合物力口上 完全弗氏佐剂免疫家兔， 15天后再用血蓝蛋白多肽复合物加不完全弗氏佐剂加强免疫 一次。 采用经 15 g/ml牛血清白蛋白多肽复合物包被的滴定板做 ELISA测定兔血清中 抗体的滴度。 用蛋白 A-Sepharose从抗体阳性的家兔血清中分离总 IgG。 将多肽结合于 溴化氰活化的 Sepharos B柱上， 用亲和层析法从总 IgG中分离抗多肽抗体。 免疫沉淀 法证明纯化的抗体可特异性地与锌指蛋白 34结合。 实施例 6: 本发明的多核苷酸片段用作杂交探针的应用
从本发明的多核苷酸中挑选出合适的寡核苷酸片段用作杂交探针有多方面的用 途， 如用该探针可与不同来源的正常组织或病理组织的基因组或 cDNA文库杂交以鉴 定其是否含有本发明的多核苷酸序列和检出同源的多核苷酸序列，进一步还可用该 探针检测本发明的多核苷酸序列或其同源的多核苷酸序列在正常组织或病理组织细 胞中的表达是否异常。 本实施例的目的是从本发明的多核苷酸 SEQ ID NO: 1中挑选出合适的寡核苷酸 片段用作杂交探针， 并用滤膜杂交方法鉴定一些组织中是否含有本发明的多核苷酸 序列或其同源的多核苷酸序列。 滤膜杂交方法包括斑点印迹法、 Southern印迹法、 Nor thern印迹法和复印方法等， 它们都是将待测的多核苷酸样品固定在滤膜上后使 用基本相同的步骤杂交。 这些相同的步骤是： 固定了样品的滤膜首先用不含探针的 杂交缓冲液进行预杂交， 以使滤膜上样品的非特异性的结合部位被载体和合成的多 聚物所饱和。 然后预杂交液被含有标记探针的杂交缓冲液替换， 并保温使探针与靶 核酸杂交。 杂交步骤之后， 未杂交上的探针被一系列洗膜步骤除掉。 本实施例利用 较高强度的洗膜条件（如较低盐浓度和较高的温度）， 以使杂交背景降低且只保留特 异性强的信号。 本实施例选用的探针包括两类： 第一类探针是完全与本发明的多核 苷酸 SEQ ID NO: 1相同或互补的寡核苷酸片段； 第二类探针是部分与本发明的多核 苷酸 SEQ ID NO: 1相同或互补的寡核苷酸片段。 本实施例选用斑点印迹法将样品固 定在滤膜上， 在较高强度的的洗膜条件下， 第一类探针与样品的杂交特异性最强而 得以保留。
一、 探针的选用
从本发明的多核苷酸 SEQ ID NO: 1中选择寡核苷酸片段用作杂交探针， 应遵循 以下原则和需要考虑的几个方面：
1， 探针大小优选范围为 18- 50个核苷酸；
2 , GC含量为 30½-70% , 超过则非特异性杂交增加；
3 , 探针内部应无互补区域；
4， 符合以上条件的可作为初选探针, 然后进一步作计算机序列分析， 包括将该初选 探针分别与其来源序列区域 （即 SEQ ID NO: 1 )和其它巳知的基因组序列及其互 补区进行同源性比较，若与非靶分子区域的同源性大于 85%或者有超过 15个连续 碱基完全相同， 则该初选探针一般就不应该使用；
5 , 初选探针是否最终选定为有实际应用价值的探针还应进一步由实验确定。
完成以上各方面的分析后挑选并合成以下二个探针：
探针 1 ( probel ), 属于第一类探针， 与 SEQ ID NO: 1的基因片段完全同源 或互补 （4 lNt ):
5'-TGTCCCTGGAACAGGAGGAGGAAACGCAACCTGGGCGGCTC-3' ( SEQ ID NO: 8 )
探针 2 ( probe2 ), 属于第二类探针， 相当于 SEQ ID NO: 1的基因片段或其 互补片段的替换突变序列 （41Nt ):
5'-TGTCCCTGGAACAGGAGGAGCAAACGCAACCTGGGCGGCTC-3' ( SEQ ID NO: 9 ) 与以下具体实验步骤有关的其它未列出的常用试剂及其配制方法请参考文献： DNA PROBES G. H. Keller; M. M. Manak; Stockton Press, 1989 (USA)以及更常用的分 子克隆实验手册书籍如《分子克隆实验指南》（1998年第二版） [美]萨姆布鲁克等著， 科学出版社。
样品制备：
1, 从新鲜或冰冻组织中提取 DNA
步骤：' 1 ) 将新鲜或新鲜解冻的正常肝组织放入浸在冰上并盛有磷酸盐缓冲液 (PBS ) 的平皿中。 用剪刀或手术刀将组织切成小块。 操作中应保持组织湿润。 2 ) 以 lOOOg 离心切碎组织 10 分钟。 3 )用冷匀浆缓冲液 ( 0.25mol/L蔗糖； 25腿 ol/L Tris-HCl, pH7.5; 25麵 ol/LnaCl; 25mmol/L MgCl2 ) 悬浮沉淀（大约 10ml/g )0 4 ) 在 4°C用电动匀浆器以全速匀浆组织悬液， 直至组织被完全破碎。 5 ) lOOOg离心 10 分钟。 6 )用重悬细胞沉淀（每 0. lg 最初组织样品加 1- 5ml ), 再以 lOOOg 离心 10 分钟。 7 )用裂解缓冲液重悬沉淀（每 O. lg最初组织样品加 lml ), 然后接以下的苯 酚抽提法。
2, DNA的苯酚抽提法
步骤： 1 )用 1- 10ml冷 PBS洗细胞， lOOOg离心 10分钟。 2 )用冷细胞裂解液重 悬浮沉淀的细胞 （Ι χ ΙΟ8细胞 /ml )最少应用 lOOul裂解缓冲液。 3 )加 SDS 至终浓 度为 1%, 如果在重悬细胞之前将 SDS直接加入到细胞沉淀中， 细胞可能会形成大的 团块而难以破碎， 并降低的总产率。 这一点在抽提 >107细胞时特别严重。 4 ) 加蛋白 酶 K至终浓度 200ug/ml。 5 ) 50°C保温反应 1小时或在 37DC轻轻振摇过夜。 6 )用等 体积苯酚： 氯仿： 异戊醇 （ 25: 24： 1 )抽提， 在小离心机管中离心 10分钟。 两相 应清楚分离， 否则重新进行离心。 7 ) 将水相转移至新管。 8 )用等体积氯仿： 异戊 醇（24: 1 )抽提， 离心 10分钟。 9 )将含 DNA的水相转移至新管。 然后进行 DNA的 纯化和乙醇沉淀。 '
3, DNA的纯化和乙醇沉淀
步骤： 1 ) 将 1八0体积 2mol/L醋酸钠和 1倍体积冷 100»/。乙醇加到 DNA溶液中， 混匀。 在- 20°C放置 1小时或至过夜。 2 )离心 10分钟。 3)小心吸出或倒出乙醇。 4 ) 用 70%冷乙醇 500ul洗涤沉淀， 离心 5分钟。 5 )小心吸出或倒出乙醇。 用 500ul冷 乙醇洗涤沉淀， 离心 5分钟。 6 )小心吸出或倒出乙醇， 然后在吸水纸上倒置使残余 乙醇流尽。 空气干燥 10-15分钟， 以使表面乙醇挥发。 注意不要使沉淀完全干燥， 否则较难重新溶解。 7 ) 以小体积 TE或水重悬 DNA沉淀。 低速涡旋振荡或用滴管吹 吸， 同时逐渐增加 TE, 混合至 DNA充分溶解， 每 1-5 X 10s细胞所提取的大约加 lui。
以下第 8-13步骤仅用于必须除去污染时， 否则可直接进行第 14步骤。
8 )将 RNA酶 A加到 DNA溶液中， 终浓度为 lOOug/ml, 37。C保温 30分钟。 9 ) 加入 SDS和蛋白酶 K, 终浓度分别为 0.5¾和 100ug/ml。 37°C保温 30分钟。 10)用等体积 的苯酚： 氯仿： 异戊醇 （ 25: 24： 1)抽提反应液， 离心 10分钟。 11)小心移出水 相， 用等体积的氯仿： 异戊醇 （24: 1) 重新抽提， 离心 10分钟。 12)小心移出水 相， 加 1/10体积 2mol/L醋酸钠和 2.5体积冷乙醇, 混匀置 - 20°C1小时。 13)用 70% 乙醇及 100»/。乙醇洗涤沉淀， 空气干燥， 重悬核酸， 过程同第 3- 6步骤。 14)测定 A26。 和 A28。以检测 DNA的纯度及产率。 15 )分装后存放于 - 20°C。
样膜的制备：
1 )取 4 X 2张适当大小的硝酸纤维素膜（NC膜）， 用铅笔在其上轻轻标出点样位置 及样号， 每一探针需两张 NC膜， 以便在后面的实验步骤中分别用高强度条件和强度 条件洗膜 。
2) 吸取及对照各 15微升， 点于样膜上， 在室温中晾干。
3 )置于浸润有 0. Imol/LNaOH, 1.5mol/LNaCl的滤纸上 5分钟 (两次）， 晾干置于 浸润有 0.5mol/L Tris-HCl (PH7.0), 3mol/LNaCl的滤纸上 5分钟 （两次）， 晾干。
4)夹于干净滤纸中， 以铝箔包好， 60-80°C真空干燥 2小时。
探针的标记
1 ) 3 μ lProbe ( 0.1OD/10 μ 1 )， 加入 2 μ IKinase缓冲液， 8-10 uCi γ- 32P-dATP+2U Kinase, 以补加至终体积 20 μ 1。
2) 37 °C 保温 2小时。
3)加 1/5体积的溴酚蓝指示剂 (ΒΡΒλ
4 ) itSephadex G-50柱。
5) 至有 P-Probe洗出前开始收集第一峰 （可用 Monitor监测）。
6) 5滴 /管， 收集 10-15管。
7)用液体闪烁仪监测同位素量
8)合并第一峰的收集液后即为所需制备的 32P- Probe (第二峰为游离 γ- 32P- dATP )。 预杂交
将样膜置于塑料袋中，加入 3- 10mg预杂交液（lOxDenhardt's; 6xSSC, 0. lmg/ml CT DM (小牛胸腺 DNA )。 ), 封好袋口后， 68°C水浴摇 2小时。
杂交
将塑料袋剪去一角， 加入制备好的探针， 封好袋口后， 42nC水浴摇过夜。
洗膜：
高强度洗膜：
1) 取出已杂交好的样膜。
2 ) 2xSSC, 0.1%SDS中， 40°C洗 15分钟 ( 2次）。 3 ) 0. lxSSC, 0. 1°/。SDS中, 40°C洗 15分钟 （ 2次）。
4 ) 0. lxSSC, 0. l»/oSDS中， 55°C洗 30分钟 （ 2次）， 室温晾干。 低强度洗膜：
1 ) 取出巳杂交好的样膜。
2 ) 2xSSC, 0. 1%SDS中 , 37°C洗 15分钟 （ 2次）。
3 ) 0. lxSSC, 0. l°/oSDS中 , 37°C洗 15分钟 （ 2次）。
4 ) 0. lxSSC, 0. 1%SDS中， 40°C洗 15分钟 （ 2次）， 室温晾干。 X-光自显影：
-70°C, X-光自显影 （压片时间根据杂交斑放射性强弱而定）。
实验结果：
采用低强度洗膜条件所进行的杂交实验， 以上两个探针杂交斑放射性强弱没有 明显区别； 而采用高强度洗膜条件所进行的杂交实验， 探针 1 的杂交斑放射性强度 明显强于另一个探针杂交斑的放射性强度。 因而可用探针 1 定性和定量地分析本发 明的多核苷酸在不同组织中的存在和差异表达。 实施例 Ί DNA Microarray
基因芯片或基因微矩阵 （DNA Microarray )是目前许多国家实验室和大制药公 司都在着手研制和开发的新技术， 它是指将大量的靶基因片段有序地、 高密度地排 列在玻璃、 硅等载体上， 然后用荧光检测和计算机软件进行数据的比较和分析， 以 达到快速、 高效、 高通量地分析生物信息的目的。 本发明的多核苷酸可作为靶 DNA 用于基因芯片技术用于高通量研究新基因功能； 寻找和筛选组织特异性新基因特别 是肿瘤等疾病相关新基因； 疾病的诊断， 如遗传性疾病。 其具体方法步骤在文献中 巳有多种报道， 如可参阅文献 DeRi s i ; J. L. , Lyer，Y. &Brown, P. 0.
(1997) Sc ience278, 680-686.及文献 Hel le, R. A. , Schema, M. , Cha i , A. , Shalom, D.， (1997) PNAS 94: 2150-2155.
(一 ) 点样
各种不同的全长 cDM共计 4000条多核苷酸序列作为靶 DNA,其中包括本发明的 多核苷酸。 将它们分别通过 PCR 进行扩增， 纯化所得扩增产物后将其浓度调到 500ng/ul左右， 用 Cartes ian 7500点样仪（购自美国 Cartes ian公司）点于玻璃介质 上， 点与点之间的距离为 280 μ ηι。 将点样后的玻片进行水合、 干燥、 置于紫外交联 仪中交联， 洗脱后干燥使 DNA固定在玻璃片上制备成芯片。 其具体方法步驟在文献 中已有多种报道， 本实施例的点样后处理步骤是：
1. 潮湿环境中水合 4小时；
2. 0.2%SDS洗涤 1分钟；
3. ddH20洗涤两次， 每次 1分钟；
4. NaBH4封闭 5分钟；
5. 95°C水中 2分钟；
6. 0.2%SDS洗涤 1分钟；
7. dd¾0冲洗两次；
8. 凉干， 25DC储存于暗处备用。
(二）探针标记 '
用一步法分别从人体混合组织与机体特定组织（或经过刺激的细胞株） 中抽提 总 mRNA, 并用 Oligotex mRNA Midi Kit (购自 QiaGen公司）纯化 mRNA,通过反转录分 另 !j将荧光 i式亦 ·] Cy3dUTP (5-Amino-propargyl-2'-deoxyuridine 5'-triphate coupled to Cy3 fluorescent dye, 购自 Amersham Pharaacia Biotech公司)标记人体混合组 织的 mRNA, 用焚光试亦 J Cy5dUTP (5-Amino-propargyl-2'-deoxyur idine 5'-triphate coupled to Cy5 fluorescent dye, 购自 Amersham Phamacia Biotech公司)标记机 体特定组织 （或经过刺激的细胞株） tnRNA， 经纯化后制备出探针。 具体步骤参照及 方法见：
Schena,
M. ,Shalon, D. , Heller, R. (1996) Proc. Natl. Acad. Sci. USA. Vol.93: 10614-10619. Sc hena,M. , Shalon, Dari. , Davis, R. W. (1995) Science.270. (20) : 467-480.
(三） 杂交
分别将来自 以上两种组织的探针与芯片一起在 UniHyb™ Hybridization
Solution (购自 TeleChem 公司）杂交液中进行杂交 16 小时， 室温用洗涤液 （ 1 x SSC, 0.2%SDS )洗涤后用 ScanArray 3000扫描仪（购自美国 General Scanning公司 ) 进行扫描， 扫描的图象用 Imagene软件（美囯 Biodiscovery公司）进行数据分析处 理， 算出每个点的 Cy3/Cy5比值。
以上机体特定组织（或经过刺激的细胞株）分别为膀胱粘膜、 PMA+的 Ecv304细胞 株、 LPS+的 Ecv304细胞株胸腺、 正常成纤维细胞 1G24NC、 Fibroblast, 生长因子刺激， 1024NT, 疤痕成 fc生长因子刺激， 1013HT、 疤痕成 fc未用生长因子刺激， 1013HC, 膀 胱癌建株细胞 EJ、 膀胱癌旁、 膀胱癌、 肝癌、 肝癌细胞株、 胎皮、 脾脏、 前列腺癌、 空肠腺癌、 贲门癌。 根据这 Π个 Cy3/Cy5比值绘出折方图。 （图 1 )。 由图可见本发明 所述的锌指蛋白 34和锌指蛋白表达谱很相似。 工业实用性
本发明的多肽以及该多肽的拮抗剂、 激动剂和抑制剂可直接用于疾病治疗， 例如， 可治疗恶性肿瘤、 肾上腺缺乏症、 皮肤病、 各类炎症、 HIV感染和免疫性 疾病等。
转录调控因子在生物体内参与决定基因在何种组织及何种发育阶段开始转录， 编码这类蛋白的基因如发生突变， 不但该基因自身不能正常表达， 而且受其调节 的许多基因也不能正常的进行转录与表达。 GATA家族是一个转录调控因子家族， 其特异的保守序列 motif 是其活性所必需。 GATA转录因子蛋白与 DNA的结合依赖 于 DNA上的一段同样的序列： （A/T)GATA(A/G)， GATA转录因子蛋白可以调控很多 基因的表达。
研究发现， GATA - 1, 它可以结合于红细胞中表达的球蛋白基因以及其他一些 基因的 GATA区域， 它对于红细胞的发育和其中基因的转录调控有重要作用； GATA- 2, 调控内皮细胞中 endothelin- 1基因的表达； GATA- 3, 可以结合于 T细胞受 体的 alpha和 delta基因的增强子， 其到调控功能； GATA- 4, 在内皮起源的组织和 心脏组织中表达。 ( Trainor CD. et al. , 1990 ) ； ( Lee ME. et al.,1991 ) ； (Ho IC. et al. , 1991); ( Spieth J. et al. ,1991 ) .
由此可见， 特异的 GATA转录因子蛋白 raotif 的表达异常， 将致使本发明的 含此 raotif 的多肽的功能异常， 从而导致很多基因表达的调控异常， 并产生相关 的疾病如红细胞疾病、 内皮组织疾病、 免疫系统疾病、 肿瘤等。
由此可见，本发明的锌指蛋白 34的表达异常将产生各种疾病尤其是红细胞疾 病、 内皮组织疾病、 免疫系统疾病、 肿瘤， 这些疾病包括但不限于：
红细胞疾病： 各种贫血如地中海贫血、 铁粒幼细胞贫血、 巨幼细胞贫血、 恶 性贫血、 再生障碍性贫血、 溶血性贫血、 骨髓病性贫血、 继发于慢性病的贫血， 红细胞增多症， 遗传性椭圆形红细胞
内皮组织疾病： 血管肉瘤， Kaposi肉瘤， 血管内皮瘤， 淋巴管瘤， 淋巴管肌 瘤， 心瓣膜病
免疫系统疾病： 原发性 T淋巴细胞免疫缺陷病如先天性胸腺发育不良、 核苷 磷酸化酶缺乏症、 慢性皮肤粘膜念球菌病、 高 IgE综合征， 原发性 B淋巴细胞免 疫缺陷病， 原发性细胞-体液联合免疫缺陷病， 原发性吞噬细胞免疫缺陷病
各种组织的肿瘤： 胃癌、 肝癌、 肺癌、 食管癌、 乳腺癌、 白血病、 淋巴瘤、 甲状腺肿瘤、 子宫肌瘤、 成神经细胞瘤、 星形细胞瘤、 室管膜瘤、 胶质细胞瘤、 结肠癌、 黑色素瘤、 肾上腺癌、 膀胱癌、 骨癌、 骨肉瘤、 骨髓瘤、 骨髓癌、 脑癌、 子宫癌、 子宫内膜癌、 胆囊癌、 结肠癌、 胸腺肿瘤、 鼻腔及鼻窦胂瘤、 鼻咽癌、 喉癌、 气管肿瘤、 纤维瘤、 纤维肉瘤、 脂肪瘤、 脂肪肉瘤、 平滑肌瘤
本发明的锌指蛋白 30的表达异常还将产生某些炎症， 某些遗传性疾病等。 本发明的多肽以及该多肽的拮抗剂、 激动剂和抑制剂可直接用于疾病治疗， 例如， 可治疗各种疾病尤其是红细胞疾病、 内皮组织疾病、 免疫系统疾病、 肿瘤， 某些炎症， 某些遗传性疾病等。
本发明也提供了筛选化合物以鉴定提高（激动剂）或阻遏（拮抗剂）锌指蛋白 34 的药剂的方法。 激动剂提高锌指蛋白 34刺激细胞增殖等生物功能， 而拮抗剂阻止 和治疗与细胞过度增殖有关的紊乱如各种癌症。 例如， 能在药物的存在下， 将哺 乳动物细胞或表达锌指蛋白 34的膜制剂与标记的锌指蛋白 34—起培养。 然后测 定药物提高或阻遏此相互作用的能力。
锌指蛋白 34的拮抗剂包括筛选出的抗体、 化合物、 受体缺失物和类似物等。 锌指蛋白 34的拮抗剂可以与锌指蛋白 34结合并消除其功能， 或是抑制该多肽的 产生， 或是与该多肽的活性位点结合使该多肽不能发挥生物学功能。
在筛选作为拮抗剂的化合物时， 可以将锌指蛋白 34加入生物分析测定中， 通 过测定化合物对锌指蛋白 34和其受体之间相互作用的影响来确定化合物是否是拮 抗剂。 用上述筛选化合物的同样方法， 可以筛选出起拮抗剂作用的受体缺失物和 类似物。 能与锌指蛋白 34结合的多肽分子可通过筛选由各种可能组合的氨基酸结 合于固相物组成的随机多肽库而获得。 筛选时， 一般应对锌指蛋白 34分子进行标 记。
本发明提供了用多肽， 及其片段、 衍生物、 类似物或它们的细胞作为抗原以生 产抗体的方法。 这些抗体可以是多克隆抗体或单克隆抗体。 本发明还提供了针对 锌指蛋白 34抗原决定簇的抗体。 这些抗体包括（但不限于）： 多克隆抗体、 单克隆 抗体、 嵌合抗体、 单链抗体、 Fab片段和 Fab表达文库产生的片段。
多克隆抗体的生产可用锌指蛋白 34直接注射免疫动物 （如家兔， 小鼠， 大鼠 等） 的方法得到， 多种佐剂可用于增强免疫反应， 包括但不限于弗氏佐剂等。 制 备锌指蛋白 34的单克隆抗体的技术包括但不限于杂交瘤技术（Kohl er and
Mi l s te in. Na ture, 1975, 256: 495-497) , 三瘤技术， 人 Β-细胞杂交瘤技术， EBV- 杂交瘤技术等。 将人恒定区和非人源的可变区结合的嵌合抗体可用已有的技术生 产（Morr i s on e t a l , PNAS , 1985 , 81 : 6851) 0 而已有的生产单链抗体的技术（U. S. Pa t No. 4946778)也可用于生产抗锌指蛋白 34的单链抗体。
抗锌指蛋白 34的抗体可用于免疫组织化学技术中，检测活检标本中的锌指蛋 白 34。 与锌指蛋白 34结合的单克隆抗体也可用放射性同位素标记， 注入体内可跟踪 其位置和分布。 这种放射性标记的抗体可作为一种非创伤性诊断方法用于肿瘤细 胞的定位和判断是否有转移。
抗体还可用于设计针对体内某一特珠部位的免疫毒素。 如锌指蛋白 34高亲和 性的单克隆抗体可与细菌或植物毒素（如白喉毒素， 蓖麻蛋白， 红豆碱等）共价结 合。 一种通常的方法是用巯基交联剂如 SPDP， 攻击抗体的氨基， 通过二硫键的交 换， 将毒素结合于抗体上， 这种杂交抗体可用于杀灭锌指蛋白 34阳性的细胞。
本发明中的抗体可用于治疗或预防与锌指蛋白 34相关的疾病。 给予适当剂量 的抗体可以刺激或阻断锌指蛋白 34的产生或活性。 . 本发明还涉及定量和定位检测锌指蛋白 34水平的诊断试验方法。 这些试验是 本领域所熟知的， 且包括 FISH测定和放射免疫测定。 试验中所检测的锌指蛋白 34 水平， 可以用作解释锌指蛋白 34在各种疾病中的重要性和用于诊断锌指蛋白 34 起作用的疾病。
本发明的多肽还可用作肽谱分析， 例如， 多肽可用物理的、 化学或酶进行特 异性切割， 并进行一维或二维或三维的凝胶电泳分析，更好的是进行质谱分析。
编码锌指蛋白 34的多核苷酸也可用于多种治疗目的。基因治疗技术可用于治 疗由于锌指蛋白 34的无表达或异常 /无活性表达所致的细胞增殖、 发育或代谢异 常。 重组的基因治疗载体 (如病毒载体）可设计用于表达变异的锌指蛋白 34 , 以抑 制内源性的锌指蛋白 34活性。 例如， 一种变异的锌指蛋白 34可以是缩短的、 缺 失了信号传导功能域的锌指蛋白 34 , 虽可与下游的底物结合， 但缺乏信号传导活 性。因此重组的基因治疗载体可用于治疗锌指蛋白 34表达或活性异常所致的疾病。 来源于病毒的表达载体如逆转录病毒、 腺病毒、 腺病毒相关病毒、 单纯疱疹病毒、 细小病毒等可用于将编码锌指蛋白 34的多核苷酸转移至细胞内。 构建携带编码锌 指蛋白 34的多核苷酸的重组病毒载体的方法可见于已有文献（Sambrook, e t a l . )。 另外重组编码锌指蛋白 34的多核苷酸可包装到脂质体中转移至细胞内。
多核苷酸导入组织或细胞内的方法包括： 将多核苷酸直接注入到体内组织中； 或在体外通过载体 (如病毒、 噬菌体或质粒等）先将多核苷酸导入细胞中， 再将细 胞移植到体内等。
抑制锌指蛋白 34 mRNA的寡核苷酸（包括反义 RNA和 DNA)以及核酶也在本发明 的范围之内。 核酶是一种能特异性分解特定 RNA的酶样 RNA分子， 其作用机制是 核酶分子与互补的靶 MA特异性杂交后进行核酸内切作用。 反义的 RM和 DNA及 核酶可用巳有的任何 RNA或 DM合成技术获得， 如固相磷酸酰胺化学合成法合成 寡核苷酸的技术巳广泛应用。 反义 RNA分子可通过编码该 RNA的 DM序列在体外 或体内转录获得。 这种 DNA序列已整合到载体的 RNA聚合酶启动子的下游。 为了 增加核酸分子的稳定性， 可用多种方法对其进行修饰， 如增加两侧的序列长度， 核糖核苷之问的连接应用磷酸硫酯键或肽键而非磷酸二酯键。
编码锌指蛋白 34的多核苷酸可用于与锌指蛋白 34的相关疾病的诊断。 编码 锌指蛋白 34的多核苷酸可用于检测锌指蛋白 34的表达与否或在疾病状态下锌指 蛋白 34的异常表达。 如编码锌指蛋白 34的 DNA序列可用于对活检标本进行杂交 以判断锌指蛋白 34的表达状况。 杂交技术包括 Sout hern印迹法， No r t he rn印迹 法、 原位杂交等。 这些技术方法都是公开的成熟技术， 相关的试剂盒都可从商业 途径得到。 本发明的多核苷酸的一部分或全部可作为探针固定在微阵列
(Mi c roa r ray)或 DMA芯片（又称为 "基因芯片" ）上， 用于分析组织中基因的差异 表达分析和基因诊断。用锌指蛋白 34特异的引物进行 RNA-聚合酶链反应（RT - PCR) 体外扩增也可检测锌指蛋白 34的转录产物。
检测锌指蛋白 34基因的突变也可用于诊断锌指蛋白 34相关的疾病。 锌指蛋 白 34突变的形式包括与正常野生型锌指蛋白 34 DNA序列相比的点突变、 易位、 缺失、 重组和其它任何异常等。 可用已有的技术如 Southern印迹法、 DNA序列分 析、 PCR和原位杂交检测突变。另外，突变有可能影响蛋白的表达，因此用 Nor the rn 印迹法、 We s tern印迹法可间接判断基因有无突变。
本发明的序列对染色体鉴定也是有价值的。 该序列会特异性地针对某条人染 色体具体位置且并可以与其杂交。 目前， 需要鉴定染色体上的各基因的具体位点。 现在， 只有很少的基于实际序列数据（重复多态性）的染色体标记物可用于标记染 色体位置。 根据本发明， 为了将这些序列与疾病相关基因相关联， 其重要的第一 步就是将这些 DNA序列定位于染色体上。
简而言之， 根据 cDNA制备 PCR引物（优选 15- 35bp) , 可以将序列定位于染色体 上。 然后， 将这些引物用于 PCR筛选含各条人染色体的体细胞杂合细胞。 只有那 些含有相应于引物的人基因的杂合细胞会产生扩增的片段。
'体细胞杂合细胞的 PCR定位法， 是将 DNA定位到具体染色体的快捷方法。 使用 本发明的寡核苷酸引物， 通过类似方法， 可利用一组来自特定染色体的片段或大 量基因组克隆而实现亚定位。 可用于染色体定位的其它类似策略包括原位杂交、 用标记的流式分选的染色体预筛选和杂交预选， 从而构建染色体特异的 cDNA库。
将 cDNA克隆与中期染色体进行荧光原位杂交（FISH) , 可以在一个步骤中精确 地进行染色体定位。 此技术的综述， 参见 Verma等， Human Chromos omes : a Manua l of Ba s i c Techni ques , Pergamon Pres s , New York (1988)。
一旦序列被定位到准确的染色体位置， 此序列在染色体上的物理位置就可以 与基因图数据相关联。这些数据可见于例如， V. Mckus i ck, Mendel i an Inher i tance in Man (可通过与 Johns Hopkins Un ivers i ty We l ch Medi ca l L i brary联机获得）。 然后可通过连锁分析， 确定基因与业已定位到染色体区域上的疾病之间的关系。
接着， 需要测定患病和未患病个体间的 cDNA或基因组序列差异。 如果在一些 或所有的患病个体中观察到某突变， 而该突变在任何正常个体中未观察到， 则该 突变可能是疾病的病因。 比较患病和未患病个体， 通常涉及首先寻找染色体中结 构的变化， 如从染色体水平可见的或用基于 cDNA序列的 PCR可检测的缺失或易位。 根据目前的物理作图和基因定位技术的分辨能力， 被精确定位至与疾病有关的染 色体区域的 cDNA, 可以是 50至 500个潜在致病基因间之一种（假定 1兆碱基作图分 辨能力和每 20kb对应于一个基因）。
- 可以将本发明的多肽、 多核苷酸及其模拟物、 激动剂、 拮抗剂和抑制剂与合 适的药物载体组合后使用。 这些载体可以是水、 葡萄糖、 乙醇、 盐类、 缓冲液、 甘油以及它们的组合。 组合物包含安全有效量的多肽或拮抗剂以及不影响药物效 果的载体和赋形剂。 这些组合物可以作为药物用于疾病治疗。
本发明还提供含有一种或多种容器的药盒或试剂盒， 容器中装有一种或多种 本发明的药用组合物成分。 与这些容器一起， 可以有由制造、 使用或销售药品或 生物制品的政府管理机构所给出的指示性提示， 该提示反映出生产、 使用或销售 的政府管理机构许可其在人体上施用。 此外， 本发明的多肽可以与其它的治疗化 合物结合使用。
药物组合物可以以方便的方式给药， 如通过局部、 静脉内、 腹膜内、 肌内、 皮下、 鼻内或皮内的给药途径。 锌指蛋白 34以有效地治疗和 /或预防具体的适应 症的量来给药。 施用于患者的锌指蛋白 34的量和剂量范围将取决于许多因素， 如 给药方式、 待治疗者的健康条件和诊断医生的判断。

Claims

杈 利 要 求 书
1、 一种分离的多肽-锌指蛋白 34， 其特征在于它包含有： SEQ ID N0: 2所示的氨 基酸序列的多肽、 或其多肽的活性片段、 类似物或衍生物。
2、 如权利要求 1所述的多肽， 其特征在于所述多肽、 类似物或衍生物的氨基酸序 列具有与 SEQ ID NO: 2所示的氨基酸序列至少 95%的相同性。
3、 如权利要求 2所述的多肽， 其特征在于它包含具有 SEQ ID NO: 2所示的氨基酸 序列的多肽。
4、 一种分离的多核苷酸， 其特征在于所述多核苷酸包含选自下组中的一种：
(a) 编码具有 SEQ ID N0: 2所示氨基酸序列的多肽或其片段、 类似物、 衍生物 的多核苷酸；
(b) 与多核苷酸 （a ) 互补的多核苷酸； 或
(c) 与 （a ) 或 （b ) 有至少 70%相同性的多核苷酸。
5、 如权利要求 4所述的多核苷酸，其特征在于所述多核苷酸包含编码具有 SEQ ID NO: 2所示氨基酸序列的多核苷酸。
6、 如权利要求 4所述的多核苷酸， 其特征在于所述多核苷酸的序列包含有 SEQ ID NO: 1中 7-924位的序列或 SEQ ID NO: 1中 1-1430位的序列。
7、一种含有外源多核苷酸的重组载体， 其特征在于它是由杈利要求 4 - 6中的任一 权利要求所述多核苷酸与质粒、 病毒或运载体表达载体构建而成的重组载体。
8、 一种含有外源多核苷酸的遗传工程化宿主细胞， 其特征在于它是选自于下列一 种宿主细胞：
(a) 用权利要求 7所述的重组载体转化或转导的宿主细胞； 或
(b) 用权利要求 4- 6中的任一权利要求所述多核苷酸转化或转导的宿主细胞。
9、 一种具有锌指蛋白 34活性的多肽的制备方法， 其特征在于所述方法包括： (a) 在表达锌指蛋白 34条件下， 培养权利要求 8所述的工程化宿主细胞； (b) 从培养物中分离出具有锌指蛋白 34活性的多肽。
10、 一种能与多肽结合的抗体,其特征在于所述抗体是能与锌指蛋白 34特异性结 合的抗体。
11、 一类模拟或调节多肽活性或表达的化合物， 其特征在于它们是模拟、 促进、 拮抗或抑制锌指蛋白 34的活性的化合物。
12、 如杈利要求 11所述的化合物， 其特征在于它是 SEQ ID NO: 1所示的多核苷酸 序列或其片段的反义序列。
1 3、 一种杈利要求 11所述化合物的应用， 其特征在于所述化合物用于调节锌指蛋 白 34在体内、 体外活性的方法。
14、 一种检测与杈利要求 1-3中的任一权利要求所述多肽相关的疾病或疾病易感 性的方法， 其特征在于其包括检测所述多肽的表达量， 或者检测所述多肽的活性， 或者检测多核苷酸中引起所述多肽表达量或活性异常的核苷酸变异。
15、 如权利要求 1-3中的任一杈利要求所述多肽的应用， 其特征在于它应用于筛 选锌指蛋白 34的模拟物、 激动剂， 拮抗剂或抑制剂； 或者用于肽指紋图谱鉴定。
16、 如权利要求 4-6中的任一杈利要求所述的核酸分子的应用， 其特征在于它作 为引物用于核酸扩增反应， 或者作为探针用于杂交反应， 或者用于制造基因芯片 或微阵列。
17、如权利要求 1-6及 11中的任一杈利要求所述的多肽、 多核苷酸或化合物的应 用， 其特征在于用所述多肽、 多核苷酸或其模拟物、 激动剂、 拮抗剂或抑制剂以 安全有效剂量与药学上可接受的载体组成作为诊断或治疗与锌指蛋白 34异常相 关的疾病的药物组合物。
18、权利要求 1-6及 11中的任一杈利要求所述的多肽、多核苷酸或化合物的应用， 其特征在于用所述多肽、 多核苷酸或化合物制备用于治疗如恶性肿瘤， 血液病， HIV感染和免疫性疾病和各类炎症的药物。
PCT/CN2001/000944 2000-06-12 2001-06-11 Nouveau polypeptide, proteine a doigt de zinc 34, et polynucleotide codant ce polypeptide WO2002000828A2 (fr)

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CN00116448.1 2000-06-12
CN 00116448 CN1327996A (zh) 2000-06-12 2000-06-12 一种新的多肽——锌指蛋白34和编码这种多肽的多核苷酸

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
EP1381621A2 (en) * 2001-03-05 2004-01-21 Hyseq, Inc. Novel nucleic acids and polypeptides

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1999062952A1 (en) * 1998-06-04 1999-12-09 Shanghai Second Medical University A human zinc finger protein gene (bmzf2)

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1999062952A1 (en) * 1998-06-04 1999-12-09 Shanghai Second Medical University A human zinc finger protein gene (bmzf2)

Non-Patent Citations (2)

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Title
DATABASE GENBANK [Online] 21 December 1999 SULSTON J.E. ET AL. Retrieved from NCBI, accession no. GI:4508146 Database accession no. (AC004890.2) *
DATABASE PROTEIN [Online] 21 October 1996 CHERET G. ET AL. Retrieved from NCBI, accession no. GI:1279698 Database accession no. (CAA61777.1) *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1381621A2 (en) * 2001-03-05 2004-01-21 Hyseq, Inc. Novel nucleic acids and polypeptides
EP1381621A4 (en) * 2001-03-05 2004-09-22 Nuvelo Inc NEW NUCLEIC ACIDS AND POLYPEPTIDES

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