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WO2002020595A1 - Nouveau polypeptide, proteine a doigt de zinc humaine 18.04, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, proteine a doigt de zinc humaine 18.04, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002020595A1
WO2002020595A1 PCT/CN2001/000937 CN0100937W WO0220595A1 WO 2002020595 A1 WO2002020595 A1 WO 2002020595A1 CN 0100937 W CN0100937 W CN 0100937W WO 0220595 A1 WO0220595 A1 WO 0220595A1
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Prior art keywords
polypeptide
polynucleotide
zinc finger
finger protein
human zinc
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PCT/CN2001/000937
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
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Priority to AU2002210314A priority Critical patent/AU2002210314A1/en
Publication of WO2002020595A1 publication Critical patent/WO2002020595A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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 18.04, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a method and application for preparing the polynucleotide and polypeptide. Background technique
  • zinc finger proteins There are a large class of proteins in the body, and their protein sequences contain one or more zinc atom binding domains. These proteins are involved in the interaction of various proteins with nucleic acids and proteins and proteins in the body. They are usually used as transcription Factors and signaling molecules are involved in the expression and regulation of related tissue genes. Such proteins constitute a large family of proteins, called zinc finger proteins. There are about 300 to 700 zinc finger proteins in the human genome. These zinc finger proteins bind to nucleotides in the body and play many important biological functions. The most important role is to regulate the transcription and expression of genes. Studies have found that zinc finger proteins are expressed in various tissues of different organisms, including hematopoietic cells, brain, nervous system, various tumor-related tissues, and tissues of immortalized cell lines. It plays an extremely important role in regulating gene transcription and expression in these tissues.
  • the zinc finger structure of zinc finger protein mainly has the following types: C2H2 configuration, C2C2 configuration, C2HC configuration, C2HC4C configuration, C3H configuration, C3HC4 configuration.
  • the zinc finger protein genes containing the C2H2 configuration constitute the largest family of genes in the human genome.
  • people have isolated zinc finger proteins of various configurations from various organisms such as yeast, fruit fly, rat and human, and carried out the biological functions of these zinc finger proteins and their chromosomal localization. Study in depth.
  • C2H2 zinc finger proteins are involved in the activation and suppression of gene transcription. These proteins are expressed differently. They often cause various developmental disorders, tumors, genetic diseases and immune system diseases [Kamal Chowdhury, Heidi Rohdegradd et al., Nucleic Acids Research, 1988, 16: 9995-10011].
  • the C2H2 zinc atom binding domain was first obtained in the protein transcription factor IIIA, and is the most widely distributed DNA binding domain in eukaryotic transcription factors.
  • the protein sequence of the C2H2 zinc finger protein contains the following conserved sequence characteristics: (Tyr, Phe) -X ⁇ Cys-X (2, 4) -Cys-X3-Phe-X5-Leu-X2- His-X ( 3,5) -His (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 Changing amino acid residues are responsible for mediating protein interactions with other molecules).
  • a C2H2 zinc finger protein may contain one or more zinc finger domains as described above, which are independent in vivo Complete your own physiological functions. In many cases, proteins containing zinc finger domains interact with special double-stranded and single-stranded DNA sequences and act as transcriptional regulators. Existing studies have found that the C2H2 zinc finger domain not only plays an important role in regulating gene expression in some tissues, but also plays a key role in the developmental regulation of organisms.
  • C2H2 zinc finger proteins can be divided into different protein families according to the similarity of their protein sequences, such as the ruppel zinc finger protein family.
  • the amino acid sequence of this protein contains five repeating and conserved MYM domains as shown below: CX2C (19-22) CX3CX (13- 19) CX2CX (19-25) FCX3CX3F / Y; This domain is an important active center for proteins that bind to the corresponding growth regulatory factor receptor proteins and play corresponding biological functions, such as proteins interacting with fibroblast growth factor receptor proteins in vivo Bind and regulate the differentiation and growth of corresponding tissue cells.
  • zinc finger protein 198 is similar to other zinc finger proteins in vivo, and it is involved in regulating the transcription and expression of various tissue genes in the body.
  • the mutation or abnormal expression of this protein will directly lead to the disorder of related tissue development and metabolism, and then cause various related diseases.
  • the protein is usually closely related to the occurrence of some nervous system disorders, developmental and metabolic disorders, hematological diseases (such as chronic leukemia), tumors of related tissues, and cancer. It can also be used to diagnose and treat various related diseases mentioned above.
  • the human zinc finger protein 18.04 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. Therefore, there has been a need in the art to identify more involved in these processes.
  • Human zinc finger protein 18.04 protein particularly the amino acid sequence of this protein. Isolation of the new human zinc finger protein 18.04 protein encoding gene also provides a basis for research to determine the role of this protein in health and disease states. This protein may constitute a disease diagnosis It is important to isolate and / or treat the drug, so it is important to isolate its coding DM. Invention, disclosure
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding human zinc finger protein 18.04.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding human zinc finger protein 18.04.
  • Another object of the present invention is to provide a method for producing human zinc finger protein 18.0.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention-human zinc finger protein 18.04.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the polypeptide of the present invention-human zinc finger protein 18..04.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities in human zinc finger protein 18.04.
  • 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 281-1775 in SEQ ID NO: 1; and (b) having a sequence of 1-1146 in SEQ ID NO: 1 Sequence of bits.
  • the present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a screening simulation, activation, antagonism or inhibition of human zinc finger protein 18.04 protein A method of active compounds, which comprises utilizing a 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 human zinc finger protein 18.04 protein in vitro, comprising detecting a mutation in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, or detecting 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 use of the polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for the treatment of cancer, developmental disease or immune disease or other diseases caused by abnormal expression of human zinc finger protein 18.04.
  • 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 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 “variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bio activity 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 human zinc finger protein 18.04, 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 human zinc finger protein 18.04.
  • Antagonist refers to a molecule that, when combined with human zinc finger protein 18.04, can block or regulate the biological or immunological activity of human zinc finger protein 18.04.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that binds human zinc finger protein 18.04.
  • Regular refers to a change in the function of human zinc finger protein 18.04, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological, functional, or immune properties of human zinc finger protein 18.04. change.
  • Substantially pure 1 means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated. 04.
  • Persons skilled in the art can purify human zinc finger protein 18.04 using standard protein purification techniques.
  • the substantially pure human zinc finger protein 18.04 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of human zinc finger protein 18.04 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence C-T-G-A
  • complementary sequence G-A-C-T.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Sou thern imprinting or Nor thern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that conditions with reduced stringency allow non-specific binding, because conditions with reduced stringency require that the two sequences bind to each other as either specific or selective interactions.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences according to different methods, such as Cluster method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). 0 Clus ter method checks all The distances arrange the groups of sequences into clusters. The clusters are then assigned in pairs or groups.
  • the percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: Number of residues matching between sequence A and sequence ⁇ 1 00
  • the number of residues in sequence A-the number of spacer residues in sequence A-the number of spacer residues in sequence B can also be determined by the Cluster method or by using known methods in the art Methods such as Jotun He in to determine the percent identity between nucleic acid sequences (He in J., (1990) Methods in emzumo l ogy 183: 625-645) 0 "Similarity" refers to the correspondence between alignments between amino acid sequences Degree of identical or conservative substitutions of amino acid residues at positions.
  • Amino acids used for conservative substitutions 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 RM sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and? ⁇ It can specifically bind to the epitope of human zinc finger protein 18.04.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not part of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances in the natural state .
  • isolated human zinc finger protein 18.04 means human zinc finger protein 18.04 is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can 04.
  • Human zinc finger protein 18.04 was purified using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human zinc finger protein 18.04 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human zinc finger protein 18.04, which basically consists of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. 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 18.04.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the human zinc finger protein 18.0 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 to another compound such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol
  • an additional amino acid sequence is fused into the mature polypeptide (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 the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a full-length polynucleotide sequence of 1146 bases, and its open reading frames 281-775 encode 164 amino acids. According to the comparison of gene chip expression profiles, it was found that this polypeptide has a similar expression profile to human zinc finger protein, and it can be inferred that the human zinc finger protein 18.04 has similar functions to human zinc finger protein.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DM 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 the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the present 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 present invention. Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity, between the two sequences).
  • the 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 a denaturant during hybridization, such as 50 ° /.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 cores. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques such as PCR to identify and / or isolate polynucleotides encoding human zinc finger protein 18.04.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding the human zinc finger protein 18.04 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 DNA isolation is the least commonly used. Direct chemical synthesis of DM sequences is often the method of choice.
  • the more commonly used method is the isolation of cDNA sequences. Isolate cDNA of interest
  • the standard method 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 proven techniques for extracting mRM. Kits are also commercially available (Qiagene).
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or loss of marker gene function; (3) determination of the transcript level of human zinc finger protein 18.04; (4) Detection of gene-expressed protein products by 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 its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is generally a DNA 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 human zinc finger protein 18.04 gene expression can be detected using 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).
  • a method for amplifying DNA / RM using PCR technology 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 DM 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 a polynucleotide of the present invention, and a host cell genetically engineered using the vector of the present invention or directly using a human zinc finger protein 18.04 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology.
  • the polynucleotide sequence encoding human zinc finger protein 18.04 can be inserted into a vector to construct Into 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 expression vector (Rosenberg, et al. Gene, 1987, 56: 125); pMSXND expression vector (Lee and Na thans, J Bi o Chem. 263: 3521, 1988) expressed in mammalian cells; and Baculovirus-derived vectors expressed in insect cells.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain origins of replication, promoters, marker genes, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human zinc finger protein 18.04 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecu lar Cloning, a Labora tory Manua, cold Harbor Labora tory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct 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, a transcription terminator, and the like. 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, polytumor 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 18.04 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.
  • 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 harvested after exponential growth phase, with (Treatment 1 2 ⁇ , with steps well known in the art. Alternatively, it is a MgCl 2. 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 liposomes Packaging, etc.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human zinc finger protein 18.04 (Science, 1984; 224: 1431). Generally there are the following steps: (1). Use the polynucleotide (or variant) encoding human human zinc finger protein 18.04 of the present invention, or transform or transduce a suitable host with a recombinant expression vector containing the polynucleotide Cell
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography
  • FIG. 1 is a comparison diagram of gene chip expression profiles of human zinc finger protein 18.04 and human zinc finger protein according to the present invention.
  • the upper graph is a graph of the expression profile of human zinc finger protein 18.04, and the lower graph is the graph of the expression profile of human zinc finger protein.
  • bladder mucosa 2- PMA + Ecv304 cell line, 3- LPS + Ecv304 cell line thymus, 4-normal fibroblasts 1024NC, 5- Fibroblast, growth factor stimulation, 1024NT, 6-scar scar fc growth factor stimulation, 1013HT, 7-scar scar into fc without stimulation with growth factor, 1013HC, 8-bladder cancer cell EJ, 9- Next to bladder cancer, 10-bladder cancer, 11-liver cancer, 12-liver cancer cell line, 13-fetal skin, 14-spleen, 15-prostate cancer, 16-jejunum adenocarcinoma, 17 cardia cancer.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of isolated human zinc finger protein 18.04. 18kDa is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention
  • Example 1 Cloning of human zinc finger protein 18.04
  • Human fetal brain total MA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using Quik mRNA I solat ion Kit (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA.
  • the Smart cDNA cloning kit purchased from Clontech was used to insert the cDNA fragments into the multicloning site of the pBSK (+) vector (Clontech) to transform DH5 ⁇ .
  • the bacteria formed a cDNA library.
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones Q667D07 was new DNA.
  • the inserted cDNA fragments contained in this clone were determined in both directions by synthesizing a series of primers.
  • the 0667D07 clone contains a full-length cDNA of 1146bp (as shown in Seq ID N0: l), and has a 494bp open reading frame (0RF) from 281bp to 775bp, encoding a new protein (such as Seq ID NO : Shown in 2).
  • This clone pBS-0667D07 and the protein encoded was human zinc finger protein 18.04.
  • Example 2 Cloning of a gene encoding human zinc finger protein 18.04 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 with Qiagene's kit, the following primers were used for PCR amplification:
  • Pr imerl 5'- GGCAGACGGTAGAGCGGAGACGAC -3 '(SEQ ID NO: 3)
  • Priraer2 5,-CAGTTCAGAAATTTTAATGCTGCT -3, (SEQ ID NO: 4)
  • Pr imerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Pr imer2 is the 3′-end reverse sequence in SEQ ID NO: 1.
  • Conditions for the amplification reaction 50 ol / L C1, 10 mmol / L Tris-CI, (pH8.5), 1.5ramol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol primers in a 50 ⁇ 1 reaction volume, 1U of Taq DNA polymerase (Clontech). Reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94 ° C 30sec; 55 C 30sec; 72 ° C 2rain 0 ⁇ -actin was set as a positive control and Template blank is negative control.
  • the amplified product was purified using a QIAGEN kit and ligated to a pCR vector (Invitrogen product) using a TA cloning kit. DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1 to 1146bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human zinc finger protein 18.04 gene expression:
  • 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% formamide-25mM KH 2 P0 4 (pH 7.4) -5 x SSC- 5 x Denhardt's solution and 200 ⁇ g / ml salmon sperm DNA. After hybridization, 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 4 In vitro expression, isolation and purification of recombinant human zinc finger protein 18.04
  • Primer3 5'-CCCCATATGATGGAAATTGCTTCGACAGGAACT-3 '(Seq ID No: 5)
  • Priraer4 5'-CATGGATCCTCACTTCCAGGCTGGCCTCCGAAG-3' (Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and BamHI restriction sites, respectively.
  • the coding sequences of the 5 'and 3' ends of the gene of interest are followed, respectively.
  • the Ndel and BamHI restriction sites correspond to the selectivity within the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Digestion site.
  • the PCR reaction was performed using pBS-0667D07 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions are as follows: a total volume of 50 ⁇ 1 contains 10 pg of pBS-0667D07 plasmid, primers Primer-3 and Primer_4, and j! (Clontech) 1 ⁇ 1. Cycle parameters: 94. C 20s, 60. C 30s, 68. C 2 min, a total of 25 cycles.
  • the amplified product and plasmid pET-28 (+) were double-digested with Mel and BamHI, respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligation product was transformed with colibacillus DH5a by the calcium chloride method.
  • the bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. The affinity chromatography column His. Bind Quick Cartridge (product of Novagen) was used to obtain 6 histidine (6His-Tag).
  • the purified human protein zinc finger protein 18.04 was obtained. After SDS-PAGE electrophoresis, a single band was obtained at 18 kDa ( Figure 2). The band was transferred to a PVDF membrane, and the N-terminal amino acid sequence was analyzed by Edams hydrolysis method. As a result, the 15 amino acids at the N-terminus were identical to the 15 amino acid residues at the N-terminus shown in SEQ ID NO: 2.
  • the following human zinc finger protein 18.04-specific peptides were synthesized using a peptide synthesizer (product of PE company): Ile-Leu- C00H (SEQ ID NO: 7).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • Rabbits were immunized with 4 mg of the above-mentioned cyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost the immunity once.
  • 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 a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods. They all use the same steps of hybridization after fixing the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer, so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthetic polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. 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 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 and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutant sequence (41Nt) of the gene fragment of 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
  • the 32 P-Probe (the second peak is free ⁇ - 32 P-dATP) is prepared after the collection solutions of the first peak are combined.
  • Gene microarray 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. , Silicon and other carriers, and then use fluorescence detection and computer software to compare and analyze the data, in order to achieve the purpose of rapid, efficient, high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases . The specific method steps have been reported in the literature.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were amplified by PCR respectively. After purification, the amplified product was adjusted to a concentration of about 500 ng / ul, and spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 ⁇ m. The spotted slide was dried in water, dried, and cross-linked in a purple diplomatic apparatus. After elution, the DM was fixed on the glass slide to prepare a chip. The specific method steps have been reported in the literature in various ways. The post-spot processing steps of this embodiment are:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and the mRNA was purified with Oligotex mRNA Midi Kit (purchased from QiaGen). The fluorescent reagent Cy3dUTP was separately reverse-transcribed.
  • mRNA of human mixed tissue was labeled with Cy5dUTP (5- Amino- propargyl- 2 ' -deoxyur idine 5'-tr iphate coupled to Cy5 fluorescent dye, purchased from Araershara Phamacia Biotech company, labeled the body's specific tissue (or stimulated cell line) mRNA, and purified the probe to prepare a probe.
  • Cy5dUTP 5- Amino- propargyl- 2 ' -deoxyur idine 5'-tr iphate coupled to Cy5 fluorescent dye, purchased from Araershara Phamacia Biotech company, labeled the body's specific tissue (or stimulated cell line) mRNA, and purified the probe to prepare a probe.
  • Solut ion (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, USA). Scanning), the scanned images were processed with Imagene software (Biodiscovery, USA) for data analysis, and the Cy3 / Cy5 ratio of each point was calculated.
  • the above specific tissues are bladder mucosa, PMA + Ecv 304 cell line, LPS + Ecv304 cell line thymus, normal fibroblasts 1024NC, Fibrobl as t, growth factor stimulation, 1024NT, scar formation fc Growth factor stimulation, 1 013HT, scar into fc without growth factor stimulation, 1013HC, bladder cancer cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunal adenocarcinoma, Cardiac cancer. Draw a graph based on these 17 Cy3 / Cy5 ratios. (figure 1 ) . It can be seen from the figure that the expression profile of human zinc finger protein 18.04 and human zinc finger protein according to the present invention are very similar. Industrial applicability
  • polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infections and immune diseases.
  • Zinc finger proteins are often used as transcription factors and signaling molecules to participate in the expression and regulation of related tissue genes. 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 immortalized cell lines.
  • C2H2 zinc finger proteins are involved in the activation and suppression of gene transcription.
  • the abnormal expression of these proteins will cause various developmental disorders, the occurrence of various tumors, various genetic diseases and immune system diseases.
  • the zinc finger protein ZNF198 is a C2H2 type zinc finger protein. It also contains a conserved MYM domain, which is an important active center of the protein that binds to the corresponding growth regulator receptor protein and exerts corresponding biological functions, such as The protein binds fibrous growth factor receptor protein in vivo and regulates the differentiation and growth of corresponding tissue cells.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of the human zinc finger protein ZNF198, and both have similar biological functions.
  • the polypeptide of the present invention is related to the transcriptional activation and inhibition of genes in the body.
  • the abnormal expression of the polypeptide usually causes abnormal gene expression in related tissues in the body, and then causes various related metabolic and developmental disorders, neurological diseases, and various related tissues. Inflammation and malignant tumors and cancers, and related diseases.
  • the abnormal expression of the human zinc finger protein 18.04 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, ependymal tumors, nodules Bowel cancer, breast cancer, leukemia, lymphoma, malignant histiocytosis, melanoma, sarcoma, myeloma, teratoma, adrenal cancer, bladder cancer, bone cancer, bone marrow cancer, brain cancer, uterine cancer, cholangiocarcinoma, liver cancer Lung cancer thymoma
  • 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
  • Abnormal expression of the human zinc finger protein 18.04 of the present invention will also produce certain genetic diseases, such as endocrine diseases, such as endocrine adenomas, and diseases of the immune system.
  • 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 for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human zinc finger protein 18.04.
  • Agonists enhance human zinc finger protein 18.04 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing human zinc finger protein 18.04 can be cultured with labeled human zinc finger protein 18.04 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human zinc finger protein 18.04 include antibodies, compounds, receptor deletions, and analogs that have been screened. Antagonists of human zinc finger protein 18.04 can bind to human zinc finger protein 18.04 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 exert its biology Features.
  • human zinc finger protein 18.04 When screening compounds as antagonists, human zinc finger protein 18.04 can be added to the bioanalytical assay In the determination, whether the compound is an antagonist is determined by measuring the effect of the compound on the interaction between human zinc finger protein 18.04 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 human zinc finger protein 18.04 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. During screening, the human zinc finger protein 18.04 molecule should generally be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies directed against the human zinc finger protein 18.04 epitope. These antibodies include (but are not limited to): Doklon antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, and fragments from Fab expression libraries.
  • Polyclonal antibodies can be produced by direct injection of human zinc finger protein 18.04 into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant.
  • Techniques for preparing monoclonal antibodies against human zinc finger protein 18.04 include, but are not limited to, hybridoma technology (ohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma technology, and EBV-hybridization Tumor technology, etc.
  • Chimeric antibodies that combine human constant regions with non-human-derived variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851).
  • the existing technology for producing single chain antibodies (U. S. Pat No. 4946778) can also be used to produce single chain antibodies against human zinc finger protein 18.04.
  • Anti-human zinc finger protein 18.04 antibodies can be used in immunohistochemistry to detect human zinc finger protein 18. 04 in biopsy specimens.
  • Monoclonal antibodies that bind to human zinc finger protein 18.04 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.
  • human zinc finger protein 18.04 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 finger protein 18.04-positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human zinc finger protein 18.04. Administration of an appropriate dose of antibody can stimulate or block the production or activity of human zinc finger protein 18.04.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human zinc finger protein 18.04. These tests are well known in the art and include FISH assays and radioimmunoassays. Tested during the test The level of human zinc finger protein 18.04 can be used to explain the importance of human zinc finger protein 18.04 in various diseases and for the diagnosis of diseases where 'broken human zinc finger protein 18.04' 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.
  • human zinc finger protein 18.04 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 18.04.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human zinc finger protein 18.04 to inhibit endogenous human zinc finger protein 18.04 activity.
  • a variant human zinc finger protein 18.04 may be a shortened human zinc finger protein 18.04 that lacks a signaling domain, although it can bind to downstream substrates, but lacks signaling activity. Therefore, recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of human zinc finger protein 18.04.
  • Expression vectors derived from viruses such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer the polynucleotide encoding human zinc finger protein 18.04 into cells.
  • viruses such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc.
  • Methods for constructing recombinant viral vectors carrying a polynucleotide encoding human zinc finger protein 18.04 can be found in the literature (Sambrook, et al.).
  • a polynucleotide encoding human zinc finger protein 18.04 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DM
  • ribozymes that inhibit human zinc finger protein 18.04 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RM to perform endonucleation.
  • Antisense RNA, DNA and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as solid-phase phosphate amide chemical synthesis technology for oligonucleotide synthesis. Widely used.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA sequence has been integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphorothioate or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human zinc finger protein 18.04 can be used for the diagnosis of diseases related to human zinc finger protein 18.04.
  • the polynucleotide encoding human zinc finger protein 18.04 can be used to detect the expression of human zinc finger protein 18.04 or abnormal expression of human zinc finger protein 18.04 in a disease state.
  • Such as encoding human zinc finger protein 18.04 The DNA sequence can be used to hybridize biopsy specimens to determine the expression of human zinc finger protein 18.04.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • a part or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray (Microarray) or a DM chip (also known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • a microarray or a DM chip (also known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Human zinc finger protein 18.04 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect human zinc finger protein 18.04 transcription products.
  • Human zinc finger protein 18.04 gene can also be used to diagnose human zinc finger protein 18.04-related diseases.
  • Human zinc finger protein 18.04 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild type human zinc finger protein 18.04 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, so Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for marking chromosome positions.
  • an important first step is to locate these DNA sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequences can be located on 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 hybrid pre-selection to construct a chromosome-specific c-band library.
  • Fluorescent in situ hybridization of cDM clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendel ian Inher i tance in Man (available via Johns Hopkins Univers i ty Welch Medica l Library available online). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with 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 invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human zinc finger protein 1 8.04 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human zinc finger protein 1 8.04 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 à doigt de zinc humaine 18.04, et un polynucléotide codant ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment de toutes sortes de tumeurs, de maladies affectant le système nerveux, des maladies sanguines malignes et des troubles du développement. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant la protéine à doigt de zinc humaine 18.04.
PCT/CN2001/000937 2000-06-12 2001-06-11 Nouveau polypeptide, proteine a doigt de zinc humaine 18.04, et polynucleotide codant ce polypeptide WO2002020595A1 (fr)

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WO1998053061A1 (fr) * 1997-05-23 1998-11-26 Amrad Operations Pty Ltd Nouveaux genes codant une proteine a doigt de zinc, un facteur d'echange de nucleotides guaniniques, une proteine du stress ou une proteine de liaison du stress
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DE4435919C1 (de) * 1994-10-07 1995-12-07 Deutsches Krebsforsch Zinkfinger-DNA, -Protein und ihre Verwendung
US5789538A (en) * 1995-02-03 1998-08-04 Massachusetts Institute Of Technology Zinc finger proteins with high affinity new DNA binding specificities
WO1998053061A1 (fr) * 1997-05-23 1998-11-26 Amrad Operations Pty Ltd Nouveaux genes codant une proteine a doigt de zinc, un facteur d'echange de nucleotides guaniniques, une proteine du stress ou une proteine de liaison du stress
WO1999021991A1 (fr) * 1997-10-29 1999-05-06 Shanghai Second Medical University Bmzf12: gene a doigt de zinc clone a partir de la moelle osseuse
WO1999045132A1 (fr) * 1998-03-02 1999-09-10 Massachusetts Institute Of Technology Proteines a poly-doigts de zinc a sequences de liaison ameliorees
WO1999046293A1 (fr) * 1998-03-12 1999-09-16 Shanghai Second Medical University Proteine a doigt de zinc derivee de cellules hematopoietiques

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