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WO2001055376A1 - Nouveau polypeptide, proteine humaine 28 a doigt de zinc contenant un motif structural hit, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, proteine humaine 28 a doigt de zinc contenant un motif structural hit, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001055376A1
WO2001055376A1 PCT/CN2001/000052 CN0100052W WO0155376A1 WO 2001055376 A1 WO2001055376 A1 WO 2001055376A1 CN 0100052 W CN0100052 W CN 0100052W WO 0155376 A1 WO0155376 A1 WO 0155376A1
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Prior art keywords
polypeptide
polynucleotide
zinc finger
finger protein
hit
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai BioDoor Gene Technology Ltd
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Shanghai BioDoor Gene Technology Ltd
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Priority to AU2001229988A priority Critical patent/AU2001229988A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide—a human zinc finger protein 28 containing a HIT structure motif, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and the polypeptide. Background technique
  • zinc finger proteins There are a wide range of proteins in living organisms, all of which contain zinc atom binding domains and are involved in protein-nucleic acid and protein-protein interactions in organisms. These zinc-binding proteins are usually involved in gene expression and regulation as transcription factors and signal transduction molecules. Such proteins constitute a large family of proteins, called zinc finger proteins. In the human genome, there are about 300 to 700 zinc finger proteins. These zinc finger proteins bind to nucleotides in the body and play many important biological functions. Their most important role is to regulate gene transcription and expression. The study found that zinc finger protein is expressed in various tissues of different organisms, including hematopoietic cells, brain, nervous system, various tumor-related tissues, and tissues of unlimited proliferation cell lines. It plays an extremely important role in the gene expression process of these tissues.
  • Zinc finger proteins can be divided into the following categories according to their zinc finger structure: C2H2, C2C2, C2HC, C2HC4C, C3H, and C3HC4 (Da i KS et a., 1998) Zinc finger proteins of various configurations have been isolated from various organisms such as Drosophila, Drosophila, rat and human, among which the C2H2 type zinc finger protein genes constitute the largest family of genes in the human genome (Berker et al., 1995). Type zinc finger proteins are involved in the activation and suppression of transcription of genes related to tissues in organisms. Abnormal expression of these proteins will cause various developmental disorders, various tumors, various genetic diseases and immune system diseases, etc. [Kama l Chowdhury, He idi Rohdekard et a l., Nucleic Ac ids Research, 1988, 16: 9995-10011] 0
  • the C2H2 zinc atom binding domain was first obtained in protein transcription factor II IA, and is the most widely distributed DNA binding domain in eukaryotic transcription factors.
  • the protein sequence of C2H2 zinc finger protein contains the following conserved sequence features: (Tyr, Phe) -X-Cys-X (2, 4) -Cy sX 3-Phe-X5-Leu-X2-H i sX ( 3, 5)-Hi s (where X represents an arbitrary amino acid residue; cysteine forms a coordination bond with histidine and a zinc atom and binds to the zinc atom; the other three conserved amino acid residues form a hydrophobic central region; Other altered amino acid residues are responsible for mediating protein interactions with other molecules).
  • a zinc finger protein may contain one or more zinc finger domains, which independently perform their own physiological functions in the body.
  • proteins containing zinc finger domains interact with special double-stranded and single-stranded DNA sequences, acting as transcription regulators. use.
  • C2H2 type zinc finger domains not only play an important regulatory role in the gene expression process of some tissues, but also play a very critical role in the developmental regulation process.
  • HIT structure motif There is also a protein family in the body.
  • the members of this family are about 12 to 14Kd, and each contains a sequence fragment composed of three histidine residues. This sequence fragment is called the HIT structure motif. Is an important area for proteins to perform normal physiological functions.
  • the HIT structure motif consists of consensus sequence fragments as shown below: [NQA] -X (4)-[GAV] -X- [QF] -X- [LIVM] -XH- [LIVMFYT]-H— [ LIVMFT] -H- [LIVMF] (2)-[PSGA]; This structural motif is combined with a zinc ion in the body to form the active conformation of the protein.
  • This structural motif is an important region where proteins bind to nucleotides to regulate gene transcription and expression in vivo. It also plays an important role in the growth and development of organisms. Mutations in this region will lead to abnormal function of the protein, which will cause diseases such as developmental disorders of various related tissues.
  • the new human zinc finger protein containing a HIT structure motif also contains a C2H2 type zinc finger domain and a HIT structure motif.
  • This protein regulates the transcription and expression of some related genes in the body, thus affecting the development of organisms.
  • the abnormal expression of this protein usually directly affects the expression of some genes in the body, that is, it is related to the occurrence of some developmental disorders of the organism, tumors of related tissues and cancer and immune system diseases. It can also be used for the diagnosis and treatment of the above diseases.
  • the human zinc finger protein 28 protein containing the HIT structural motif plays an important role in important functions in the body, 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 involved in these processes.
  • Human zinc finger protein 28 protein containing the HIT structural motif in particular the amino acid sequence of this protein is identified. Isolation of the zinc finger protein 28 protein gene containing the HIT structural motif by the newcomers also provides a basis for studying the role of this protein in health and disease states. This protein may form the basis for developing diagnostic and / or therapeutic drugs for diseases, so isolating its coding DNA is important. Disclosure of invention
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding human zinc finger protein 28 containing a HIT structural motif.
  • Another object of the present invention is to provide a method for producing human zinc finger protein 28 containing a HIT structural motif. Another object of the present invention is to provide an antibody against the polypeptide of the present invention-human zinc finger protein 28 containing a HIT scab motif.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors of the zinc finger protein 28 containing the HIT structure motif of the polypeptide of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities in zinc finger protein 28 containing a human HIT structural motif.
  • 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 311 to 1075 in SEQ ID NO: 1; and (b) a sequence having 1-1595 in SEQ ID NO: 1 Sequence of bits.
  • the invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • 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 human zinc finger protein 28 protein containing the HIT structure motif, which comprises utilizing the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for in vitro detection of a disease or disease susceptibility related to abnormal expression of a human HIT structural motif-containing zinc finger protein 28 protein. Mutates, or detects the amount or biological activity of a polypeptide of the invention in a biological sample.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the preparation of a polypeptide and / or polynucleotide of the present invention for the treatment of cancer, developmental disease or immune disease or other medicines caused by abnormal expression of zinc finger protein 28 containing HIT structural motif in human. use.
  • Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein.
  • the following terms used in this specification and claims have the following meanings unless specifically stated: "Nucleic acid sequence" refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a 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 means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response in appropriate animals or cells and to bind to specific antibodies.
  • An "agonist” refers to a molecule that, when combined with a human zinc finger protein containing a HIT structural motif, can cause the protein to change, thereby regulating the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to a human HIT-containing motif zinc finger protein 28.
  • Antagonist refers to a biological activity or immunity that can block or regulate human zinc finger protein 28 containing HIT structure motif when combined with human zinc finger protein 28 containing HIT structure motif.
  • Chemically active molecules can include proteins, nucleic acids, carbohydrates, or any other molecule that can bind human zinc finger protein 28 containing a HIT-containing motif.
  • Regular refers to a change in the function of human zinc finger protein 28 containing a HIT structural motif, including protein activity Increase or decrease in sexuality, changes in binding characteristics, and any other biological, functional, or immune properties of human HIT-structure-containing zinc finger protein 28.
  • substantially pure is meant substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify human zinc finger protein containing HIT structural motifs using standard protein purification techniques.
  • the substantially pure human zinc finger protein containing the HIT structure motif 28 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of human zinc finger protein 28 peptide containing HIT structure motif can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of a nucleotide by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence "C-T-G-A” can be combined with the complementary sequence "G-A-C-T”.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern blotting or Nor thern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other specifically or selectively.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as through the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). 0 The MEGALIGN program can compare two or more sequences according to different methods such as the Clus ter method (Hi gg ins, DG and PM Sharp (1988) Gene 73: 237-244). 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:
  • nucleic acid sequences can also be determined by the Cluster method or by a method known in the art such as Jotun He in. Percentages (Hein J., (1990) Methods in emzumology 183: 625-645).
  • Similarity refers to the identity or preservation of amino acid residues at corresponding positions in the alignment of amino acid sequences. The extent of conservative substitution.
  • Amino acids used for conservative substitutions for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to the “sense strand”.
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. Such a chemical modification may be a substitution of a hydrogen atom with a fluorenyl group, an acyl group or an amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological characteristics of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and?, which can specifically bind to the epitope of human zinc finger protein 28 containing the HIT structural motif.
  • 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 human HIT structure motif-containing zinc finger protein 28 means that the human HIT structure motif-containing zinc finger protein 28 is substantially free of other proteins, lipids, sugars, or Other substances. Those skilled in the art can use standard protein purification techniques to purify human zinc finger proteins containing the HIT structure motif. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human zinc finger protein 28 peptide containing the H IT structure motif can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide—a human zinc finger protein 28 containing a HIT structure motif, 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 polypeptide of the present invention may be a naturally purified product, or It is produced synthetically, or is produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant technology. 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 human zinc finger protein 28 containing a HIT structural motif.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human HIT-containing zinc finger protein 28 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 type 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
  • (IV) a type in which the additional amino acid sequence is fused into the mature polypeptide and the polypeptide sequence is formed (Such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protease sequence)
  • such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes a nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 1959 bases in length and its open reading frames 311-1075 encode 254 amino acids.
  • This polypeptide has a characteristic sequence of a C2H2 zinc finger domain and a HIT structure motif. It can be inferred that the human zinc finger protein 28 containing a HIT structure motif has a structure represented by a C2H2 zinc finger domain and a HIT structure motif.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide is meant to include polynucleotides that encode such polypeptides and polynucleotides that include additional coding and / or noncoding 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 (with at least two sequences between
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • "strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficol 1, 42 ° C, etc .; or (3) only between the two sequences Hybridization occurs only when the identity is at least 95%, and more preferably 97%.
  • the hybridizable polynucleotide-encoded polypeptide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nuclei. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding human zinc finger protein 28 containing a HIT structure motif.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence of the present invention encoding human HIT-containing motif-containing zinc finger protein 28 can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DM sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the separation of the CDM sequences.
  • the standard method for isolating cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature techniques for mRNA extraction, and kits are also commercially available (Q i agene). And the construction of cDNA libraries is also a common method (Sambrook, et al., Molecule Laring, A Labora tory Manua, Colling Harbor Labora tory. New York, 1989). Commercially available cDNA libraries, such as different cDNAs from Clontech library. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DM-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of human transcripts of zinc finger protein 28 containing HIT structural motifs (4) Detecting the protein product of gene expression by immunological techniques or measuring biological activity. The above methods can be used alone or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably Is at least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is usually a DM sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of human zinc finger protein 28 gene containing HIT structural motifs can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). .
  • a method (Saiki, et al. Science 1985; 230: 1350-1 354) using DNA technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, the sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a human HIT structure motif-containing zinc finger protein 28 coding sequence, and recombinant technology to produce the present invention Methods of the polypeptide.
  • a polynucleotide sequence encoding a human zinc finger protein-containing zinc finger protein 28 motif can be inserted into a vector to form 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 Virus, retrovirus or other vector.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors expressed in bacteria (Rosenberg, et al. Gene, 1987, 56: 125); pMSXND expression vectors expressed in mammalian cells ( Lee and Nathans, J Bio Chera.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods well known to those skilled in the art can be used to construct an expression vector containing a DM sequence encoding a human HIT-containing structural motif zinc finger protein 28 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells.
  • Enhancers are cis-acting factors expressed by DM, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, tumorigenic 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 selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human zinc finger protein 28 containing a HIT structure motif or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetic engineering containing the polynucleotide or the recombinant vector.
  • Host cells refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • Animal cells insect cells
  • fly S2 or Sf 9 animal cells
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of DNA uptake can be in the exponential growth phase were harvested, treated with CaC l 2 method used in steps well known in the art. The alternative is to use MgC l 2 .
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human HIT structure motif-containing zinc finger protein 28 (Scence, 1984; 224: 1431). Generally speaking, there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography
  • FIG. 1 is a comparison diagram of the amino acid sequences of the zinc finger protein 28 and C2H2-type zinc finger domains and HIT structure motifs containing the H IT structure motif of the present inventors.
  • FIG 2 is a HIT structure containing isolated human zinc finger motifs polyacrylamide gel electrophoresis (SDS-PAGE) 0 28KDa protein is a protein of molecular weight 28. The arrow indicates the isolated protein band. The best way to implement the invention
  • RNA Human fetal brain total RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using Quik mRNA Isolat ion Kit (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA.
  • the Smart cDNA cloning kit purchased from Clontech
  • the bacteria formed a cDNA library.
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the inserted cDNA fragment contained in this clone was determined in both directions by synthesizing a series of primers.
  • the 0582d08 clone contained a full-length cDNA of 1959bp (as shown in Seq ID NO: 1), and a 765bp open reading frame (0RF) from 311bp to 1075bp, encoding a new protein (such as Seq ID NO : Shown in 2).
  • This clone pBS-0582d08 and encoded the protein as a human zinc finger protein containing a HIT structure motif.
  • Example 2 Domain analysis of cDNA clones
  • the sequence of the human zinc finger protein 28 containing the HIT structure motif of the present invention and the protein sequence encoded by the same were applied to the profiling scan tool (BasiclocalAlignment search tool) in GCG [Al tschul, SF et al. J Mol. Biol. 1990; 215: 403-10], domain analysis was performed in databases such as Prote.
  • the human HIT structure-containing zinc finger protein 28 of the present invention is homologous to the domain C2H2 type zinc finger domain and the HIT structure motif. The results of the homology are shown in FIG. 1.
  • Example 3 Cloning of a gene encoding human zinc finger protein 28 containing HIT motif by RT-PCR CDNA was synthesized using fetal brain cell total RNA as a template and ol igo-dT as a primer for reverse transcription reaction. After purification using Qiagene's kit, the following primers were used for PCR amplification:
  • Primerl 5'- GAGGCACAGATGAGTAACGTGAAT -3 '(SEQ ID NO: 3)
  • Primer2 5'- ACTGTTTTATCCAAATTTATTCTC -3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp of the 5th end of SEQ ID NO: 1;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification reaction conditions 50 mmol / L C1, 10 mmol / L Tris-CI, (pH 8.5), 1.5 mmol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol primer, 1U in a reaction volume of 50 ⁇ 1 Taq DNA polymerase (C 1 on tech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perk in-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min.
  • ⁇ -actin was set as a positive control and template blank was set as a negative control.
  • 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 l-1959bp shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of human zinc finger protein 28 gene containing HIT structural motifs:
  • RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] 0
  • This method involves acid guanidinium thiocyanate-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 time volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ), Mix and centrifuge. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • RNA was synthesized by electrophoresis on a 1.2% agarose gel containing 20raM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 mM EDTA-2.2 M formaldehyde. It was then transferred to a nitrocellulose membrane.
  • the DM probe used was the human HIT structure motif-containing zinc finger protein 28 coding region sequence (311bp to 1075bp) amplified by PCR as shown in FIG. 1.
  • a 32P-labeled probe (approximately 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50 ° /.
  • the filter was washed in 1 x SSC-0.1% SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 5 In vitro expression, isolation and purification of recombinant human HIT structure motif-containing zinc finger protein 28
  • the Nhel and BamHI restriction sites correspond to the selective endonucleases on the expression vector plasmid pET 28b (+) (Novagen, Cat. No. 69865.3). Enzyme site.
  • PCR was performed using the pBS-0582d08 plasmid containing the full-length target gene as a template. The PCR reaction conditions were: pBS-0582d08 plasmid 10pg, 5
  • Nhel and BamHI were used to double-digest the amplified product and plasmid P ET-28 (+), respectively, and large fragments were recovered respectively and ligated with T4 ligase.
  • the ligation product was transformed into E. coli DH5a by the calcium chloride method. After being cultured overnight on an LB plate containing kanamycin (final concentration 30 g / ml), positive clones were selected by colony PCR and sequenced. A positive clone (pET-0582d08) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) by the calcium chloride method.
  • the host strain BL21 (pET-0582d08) was at 37 in LB liquid medium containing kanamycin (final concentration 30 M g / ml). C. Cultivate to logarithmic growth phase, add IPTG to a final concentration of 1 mmol / L, and continue incubating for 5 hours. The bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. The affinity chromatography column His. Bind Quick Cartridge (product of Novagen) was used for chromatography to obtain 6 histidines (6His-Tag). The purified human protein Zinc finger protein 28 containing HIT motif was described.
  • Polypeptide synthesizer (product of PE company) was used to synthesize the following human HIT-containing motif zinc finger protein 28-specific peptides:
  • a titer plate coated with a 15 M g / ml bovine serum albumin peptide complex was used as an ELISA to determine the antibody titer in rabbit serum.
  • Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose.
  • the peptide was bound to a cyanogen bromide-activated Sepharose4B column, and the anti-peptide was separated from the total IgG by affinity chromatography.
  • Antibodies The immunoprecipitation method proved that the purified antibody could specifically bind to human zinc finger protein 28 containing HIT structural motif.
  • Example 7 Use of a polynucleotide fragment of the present invention as a hybridization probe
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern imprinting, Nor thern blotting, and copying methods. They all use the same steps to fix the polynucleotide sample to be tested on the filter and then hybridize.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature), so that the hybridization background is reduced and only strong specific signals are retained.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • 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 for use as hybridization probes from the polynucleotide SEQ ID NO: 1 of the present invention should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • the GC content is 30% -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 known genomic sequences Compare its homology with its complementary region. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used in general;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 which belongs to the second type of probe, is equivalent to the replacement mutant sequence of the gene fragment or its complementary fragment (41Nt) of SEQ ID NO: 1
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • Two NC membranes are required for each probe for subsequent experiments.
  • the film is washed with high-strength conditions and strength conditions, respectively.
  • the 32 P-Probe (the second peak is free ⁇ - 32 P-dATP) is prepared after the collection solutions of the first peak are combined.
  • probe 1 can be used for qualitative and quantitative analysis.
  • the presence and differential expression of the polynucleotide of the present invention in different tissues are analyzed.
  • 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 large numbers of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as 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 DeRisi, JL, Lyer, V. & Brown, P.0. (1997) Science 278, 680-686. And the literature Helle, RA, Schema, M. , Chai, A., Shalom, D., (1997) PNAS 94: 2150-2155.
  • a total of 4,000 polynucleotide sequences of various full-length cDMs are used as target DNA, including the polynucleotide of the present invention. They were amplified by PCR respectively. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and spotted on a glass medium with a Cartesian 7500 spotting instrument (purchased from Cartesian, USA). The distance is 280 ⁇ . The spotted slides were hydrated, dried, and cross-linked in a UV cross-linking instrument. After elution, the DNA was fixed on the glass slide to prepare a chip. The specific method steps have been variously reported in the literature. The post-spotting processing steps of this embodiment are:
  • Total mRNA was extracted from normal liver and liver cancer in one step, and the mRNA was purified using Oligotex mRNA Midi Kit (purchased from QiaGen).
  • the fluorescent reagent Cy3dUTP (5- Amino- propargyl-2'-deoxyur idine) was reverse-transcribed separately. 5'-triphate coupled to Cy3 fluorescent dye, purchased (Amersham Phamacia Biotech) labeled normal liver tissue mRNA, labeled with Cy5dUTP (5-Amino-propargyl-2'-deoxyur idine 5'-triphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech), labeled liver cancer mRNA The probe was prepared after purification. For specific steps and methods, see:
  • the probes from the two types of tissues and the chip were hybridized in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, washed with a washing solution (lx SSC, 0.2 SDS) at room temperature, and then scanned with a ScanArray 3000 scanner. (Purchased from General Scanning Company, USA) for scanning, and the scanned image was analyzed with Imagene software (Biodiscovery Company, USA) to calculate the Cy3 / Cy5 ratio of each point, and the point with the ratio less than 0.5 and greater than 2 was considered as expression Different genes.
  • the polypeptides of the present invention 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.
  • Zinc-binding protein is usually used as a transcription factor and signal transduction molecule to participate in gene expression and regulation.
  • Zinc finger protein is expressed in blood cells, brain, nervous system, various tumor-related tissues, and tissues of immortal cell lines, including C2H2 Proteins of type zinc finger domain also play a key role in the developmental regulation process.
  • C2H2 type zinc finger domains are related to the following diseases: solid tumors such as thyroid adenoma, uterine fibroids, neurological diseases such as extrapyramidal dysfunction, Parkinson's syndrome, ataxia, nerve cells Tumors, glioblastomas, hematological malignancies such as leukemia, non-Hodgkin's lymphoma, developmental disorders such as Williams Syndrome, split-foot and cracked feet, Bayesian Syndrome, other tumors such as neuroblastoma , Colon cancer, breast cancer and more.
  • solid tumors such as thyroid adenoma, uterine fibroids
  • neurological diseases such as extrapyramidal dysfunction, Parkinson's syndrome, ataxia
  • nerve cells Tumors such as leukemia, non-Hodgkin's lymphoma
  • developmental disorders such as Williams Syndrome, split-foot and cracked feet
  • Bayesian Syndrome other tumors such as neuroblastoma , Colon cancer, breast cancer and more.
  • the polypeptide of the present invention is a C2H2 type zinc finger protein, and it also contains a characteristic HIT structure motif.
  • This structural motif is an important region where proteins bind to nucleotides to regulate gene transcription and expression in vivo. It can be seen that the abnormal expression of the zinc finger protein 28 containing the HIT structural motif of the present invention will produce various diseases, especially various tumors, neurological diseases, hematological malignant diseases, and developmental disorders. These diseases include, but are not Limited to:
  • Tumors of various tissues thyroid tumors, uterine fibroids, neuroblastomas, ependymoma, colon cancer, breast cancer, leukemia, lymphoma, malignant histiocytosis, melanoma, sarcoma, myeloma, teratoma , Adrenal cancer, bladder cancer, bone cancer, bone marrow cancer, brain cancer, uterine cancer, gallbladder cancer, liver cancer, lung cancer, thymic tumor
  • Nervous system diseases neural tube insufficiency such as spina bifida, anencephaly malformation, brain (meningeal) bulge, craniocerebral fissure, neural tube cysts, brain developmental abnormalities such as foramen malformations, tetanus, hydrocephalus, neuronal migration Obstacles such as abnormal formation of the brain gyrus, other malformations such as aqueduct malformations, cerebellar dysplasia, Down syndrome, spinal deformity, congenital hydrocephalus, congenital cerebral nucleus dysplasia syndrome, glioma, meningiomas, Neurofibromas, Pituitary Adenomas, Intracranial Granuloma, Alzheimer's Disease, Parkinson's Disease, Dance, Depression, Amnesia, Huntington's Disease, Epilepsy, Migraine, Dementia, Multiple Sclerosis, Mental Schizophrenia, depression, paranoia, anxiety, obsessive-compulsive disorder
  • Hematological malignancies Leukemia, non-Hodgkin's lymphoma
  • the abnormal expression of the human zinc finger protein 28 containing the HIT structural motif of the present invention will also produce certain genetic diseases, endocrine system diseases such as endocrine adenoma, and immune system diseases.
  • 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 various tumors, nervous system diseases, hematological malignant diseases, development disorders, etc. Some genetic diseases, endocrine system diseases such as endocrine adenoma, immune system diseases, etc.
  • the invention also provides methods of screening compounds to identify agents that increase (agonist) or suppress (antagonist) human HIT structural motif-containing zinc finger protein 28. Agonists enhance human zinc finger protein 28 containing the HIT structural motif to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • mammalian cells or human HIT-containing cells can be expressed in the presence of drugs.
  • the membrane preparation of the zinc motif protein 28 with the structural motif was cultured together with the labeled human zinc finger protein 28 containing the HIT structural motif. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human zinc finger protein 28 containing the H IT structure motif include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human zinc finger protein 28 containing the HIT structural motif can bind to human zinc finger protein 28 containing the HIT structural motif and eliminate its function, or inhibit the production of the polypeptide, or with the active site of the polypeptide Dot binding prevents the polypeptide from functioning biologically.
  • human zinc finger protein 28 containing the H IT structural motif can be added to the bioanalytical assay. The effect of this interaction is used to determine whether the compound is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same way as for screening compounds described above.
  • Polypeptide molecules capable of binding to human zinc finger protein 28 containing a HIT structural motif 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 human zinc finger protein containing HIT structure motif should generally be labeled with 28 molecules.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against human zinc finger protein 28 epitopes containing the HIT structural motif. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments generated from Fab expression libraries.
  • Polyclonal antibodies can be produced by direct injection of human HIT-containing motif zinc finger protein 28 into immunized animals
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant.
  • Techniques for preparing human monoclonal antibodies containing zinc finger protein 28 with HIT structural motifs include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), three tumor technology, human B-cell hybridoma technology, EBV-hybridoma technology, etc. Chimeric antibodies that bind human constant regions and non-human variable regions can be produced using existing techniques (Morri son et al, PNAS, 1985, 81: 6851). 0 Existing techniques for producing single-chain antibodies (US Pa t No. 4946778) can also be used to produce single chain antibodies against human HIT structure motif-containing zinc finger protein 28.
  • Anti-human HIT structure motif-containing zinc finger protein 28 can be used in immunohistochemical techniques to detect human HIT structure motif-containing zinc finger protein 28 in biopsy specimens.
  • Monoclonal antibodies that bind to human zinc finger protein 28 containing H IT structural motifs can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis. Antibodies can also be used to design immunotoxins that target a particular part of the body. For example, human HIT-containing zinc finger protein 28 high affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human zinc fingers containing HIT structure motifs Protein 28 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human zinc finger protein 28 containing a HIT structural motif.
  • Administration of appropriate doses of antibodies can stimulate or block the production or activity of human zinc finger protein 28 containing HIT structural motifs.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of zinc finger protein 28 in humans containing the HIT structural motif.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human zinc finger protein 28 containing HIT structural motifs detected in the test can be used to explain the importance of human zinc finger protein 28 containing HIT structural motifs in various diseases and to diagnose human HIT structural motifs A disease in which zinc finger protein 28 functions.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • Polynucleotides encoding human zinc finger protein 28 containing a HIT structural motif can also be used for a variety of therapeutic purposes.
  • Gene therapy technology can be used to treat abnormal cell proliferation, development or metabolism caused by the non-expression or abnormal / inactive expression of human zinc finger protein 28 containing HIT structural motifs.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human HIT structure motif-containing zinc finger protein 28 to inhibit endogenous human HIT structure motif-containing zinc finger protein 28 activity.
  • a variant human zinc finger protein 28 containing a HIT structural motif may be a shortened human zinc finger protein 28 containing a HIT structural motif that lacks a signaling domain, although it can bind to a downstream substrate, However, 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 28 containing HIT structural motifs in humans.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding human zinc finger protein 28 containing a HIT structural motif into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a human HIT-containing motif zinc finger protein 28 can be found in the existing literature (Sambrook, et al.).
  • a polynucleotide encoding human zinc finger protein 28 containing a HIT structural motif 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 through a vector (such as a virus, phage, or plasmid) in vitro, The cells are then transplanted into the body and the like.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit human zinc finger protein 28 mRNA containing HIT structural motifs are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA and D and ribozymes can be obtained by any existing RM or DNA synthesis technology. For example, solid-phase phosphoramidite chemical synthesis technology has been widely used for oligonucleotides.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of the DM sequence encoding the RM. This DNA sequence has been integrated downstream of the RM polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human zinc finger protein 28 with HIT structure motif can be used for diagnosis of diseases related to human zinc finger protein 28 with HIT structure motif.
  • the polynucleotide encoding human zinc finger protein 28 containing HIT structure motif can be used to detect the expression of human zinc finger protein 28 containing HIT structure motif or human zinc finger protein 28 containing HIT structure motif in a disease state Abnormal expression.
  • the DNA sequence encoding human zinc finger protein 28 containing HIT structural motif can be used to hybridize biopsy specimens to determine the expression status of human zinc finger protein 28 containing HIT structural motif.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization.
  • RNA-polymerase chain reaction (RT-PCR) amplification using human HIT-containing motif zinc finger protein 28-specific primers can also be used to detect human HIT-containing motif zinc finger protein 28 transcription products.
  • Detection of mutations in the zinc finger protein 28 gene containing human HIT structural motifs can also be used to diagnose human zinc finger protein 28-containing diseases containing HIT structural motifs.
  • Human HIT structure motif-containing zinc finger protein 28 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type human HIT structure motif-containing zinc finger protein 28 DM sequences. 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, Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for labeling chromosome positions.
  • it The important first step is to locate these DNA sequences on the chromosome.
  • PCR primers (preferably 15-35bp) are prepared from the cDNA, and the sequences can be located on the chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization (FI SH) of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FI SH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckus ck, Mendel ian Inher tance in Man (available online with Johns Hopkins University Wetch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with 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 which 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.
  • containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • instructional instructions given by government regulatory agencies that manufacture, use, or sell pharmaceuticals or biological products, which instructions reflect production, use Or a government agency that sells it allows it to be administered to humans.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human zinc finger protein containing a HIT structural motif is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of zinc finger protein 28 containing the HIT structural motif in a human being administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine humaine 28 à doigt de zinc contenant un motif structural HIT, et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la protéine humaine 28 à doigt de zinc contenant un motif structural HIT.
PCT/CN2001/000052 2000-01-26 2001-01-15 Nouveau polypeptide, proteine humaine 28 a doigt de zinc contenant un motif structural hit, et polynucleotide codant pour ce polypeptide Ceased WO2001055376A1 (fr)

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CN 00111521 CN1307024A (zh) 2000-01-26 2000-01-26 一种新的多肽——人含hit结构基序的锌指蛋白28和编码这种多肽的多核苷酸

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US7119186B2 (en) * 2001-09-14 2006-10-10 Athena Diagnostics, Inc. Application of aprataxin gene to diagnosis and treatment for early-onset spinocerebellar ataxia (EAOH)

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CN110527727B (zh) * 2019-07-05 2021-10-29 江苏医药职业学院 检测锌指蛋白28表达水平的试剂的应用和试剂盒

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US7119186B2 (en) * 2001-09-14 2006-10-10 Athena Diagnostics, Inc. Application of aprataxin gene to diagnosis and treatment for early-onset spinocerebellar ataxia (EAOH)
US7824860B2 (en) 2001-09-14 2010-11-02 Athena Diagnostics, Inc. Application of aprataxin gene to diagnosis and treatment for early-onset spinocerebellar ataxia (EAOH)

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CN1307024A (zh) 2001-08-08

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