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WO2001055379A1 - Nouveau polypeptide, proteine humaine 48 d'inhibition de la croissance cellulaire, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, proteine humaine 48 d'inhibition de la croissance cellulaire, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001055379A1
WO2001055379A1 PCT/CN2001/000076 CN0100076W WO0155379A1 WO 2001055379 A1 WO2001055379 A1 WO 2001055379A1 CN 0100076 W CN0100076 W CN 0100076W WO 0155379 A1 WO0155379 A1 WO 0155379A1
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Prior art keywords
polypeptide
polynucleotide
protein
human
sequence
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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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    • 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

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, human cell growth inhibitory protein 48, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique
  • Type I protein is a transmembrane protein that plays an important regulatory role in the development of the organism and the immune system, and regulates the development and growth of relevant tissues in the body. This type of protein is encoded and expressed by a multi-gene family, and its expression is controlled by some regulatory factors. The abnormal expression of this protein is usually related to the development of some developmental disorders and autosomal immunodeficiency diseases.
  • Type A defective factor is also known as CI ITA; Type C is transcription factor 5 (RFX5); and Type D defective is the X chromosome-related regulatory factor protein RFXAP.
  • CI ITA is a non-DM binding regulator, which regulates the morphological specificity of cells and the expression of various cells in vivo.
  • RFX5 and RFXAP are two subunits of regulatory factor (RFX), a multi-protein complex that binds to the histocompatibility protein X-box structure motif.
  • RFXANK is also an ankyrin-like protein, the protein sequence of which contains a protein-protein interaction region, which also contains three ankyrin repeat sequence fragments, each ankyrin repeat sequence domain contains a spiral- ⁇ fold -Helix secondary structure, this domain is the active center of protein action, and its mutation will directly lead to the abnormal action of protein. Proteins containing this domain are usually closely related to the occurrence of some common hereditary hemolytic anemia in the body. [Lux SE, Tse WT et a l., 1990, Nature, 345: 736-9].
  • This protein subunit is responsible for the interaction with RFX5 and RFXAP in the body. It is necessary for the RFX complex to bind with the histocompatibility type II protein promoter and regulate the role of histocompatibility type II protein. E. et al., 1998, Nat Genet, 20: 273-7].
  • Uroporphyrinogen decarboxylase is the fifth enzyme in the heme biosynthetic pathway and catalyzes the decarboxylation of the four acylation sites of uroporphyrinogen to form fecal porphyrinogen.
  • Different members of the family have been isolated from various tissues, including human, chicken red blood cells, and cattle liver. Studies have found that members of the enzyme family are highly expressed in tissues related to blood cell metabolism, and they are involved in important metabolic processes such as blood cell generation in related tissues in the body. Cheng. Deletion of uroporphyrinogen decarboxylase is usually closely related to the occurrence of some family hereditary diseases in humans, such as: fPCT and hepatic hematopoiesis, etc. [Garey JR, Labbe-Boi s R. et a l., 1992, Eur. J. Biochem. 205: 1011-1016] 0
  • the protein sequence of uroporphyrinogen decarboxylase is evolutionarily highly conserved.
  • the N-terminus of the protein family member protein sequence contains a highly conserved region, which is a characteristic sequence fragment of the enzyme family.
  • Another characteristic sequence fragment of this enzyme family is a conserved region located in the central region of the protein.
  • Sequence fragment 1 PXWXMRQAGR
  • Sequence fragment 2 GF- [STAGCV]-[STAGC] -XP- [FYW ] -T- [LV] -X (2) -YX (2)-[AE]-[G]
  • Sequence fragment 1 contains a six-petal peptide chain structure, and this petal structure contains two arginines Residues, they are bound to the propionate carboxyl group of the substrate through the salt bridge, so the characteristic sequence fragment is the central region of the enzyme to perform normal physiological functions. Mutations in this region will cause the protein to function abnormally, affecting the carboxyl group in the metabolic process. Metastasis, which causes various related diseases.
  • the novel human cytostatic protein of the present invention also contains the ankyrin repeating sequence fragment domain of the above-mentioned compatibility class II protein expression regulator subunit 3 and characteristic sequence fragments of the uroporphyrinogen decarboxylase family
  • the two domains are related to the occurrence of various blood system diseases and immunodeficiency diseases in vivo.
  • the human gene of the present invention is a new human cell growth inhibitory protein. It is also related to the occurrence of various diseases of the blood system in the body.
  • the protein is usually associated with some hereditary blood system diseases, such as hemolytic anemia, hepatocyte hematopoietic purpura, etc .; tumors and cancer of some related tissues;
  • the occurrence of type I diseases is closely related. It can also be used to diagnose and treat the above-mentioned related diseases.
  • human cytostatic protein 48 plays an important role in important functions in the body as described above, and it is believed that a large number of proteins are involved in these regulatory processes, there has been a need in the art to identify more human cytostatic proteins involved in these processes. 48 proteins, especially the amino acid sequence of this protein. Isolation of the new human cytostatic protein 48 protein-coding gene also provided the basis for the study to determine 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 cytostatic protein 48.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human cytostatic protein 48.
  • Another object of the present invention is to provide a method for producing human cytostatic protein 48.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention-human cytostatic protein 48.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the polypeptide of the present invention-human cytostatic protein 48.
  • Another object of the present invention is to provide a method for diagnosing and treating a disease associated with an abnormality of human cytostatic protein 48.
  • 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 97-1404 in SEQ ID NO: 1; and (b) a sequence having positions 1-15 in SEQ ID NO: 1 36-bit sequence.
  • 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 cytostatic protein 48 protein, which comprises utilizing the polypeptide of the invention.
  • the present invention also relates to obtaining the method.
  • the present invention also relates to a method for detecting a disease or disease susceptibility related to abnormal expression of human cell growth inhibitory protein 48 in vitro, which comprises detecting the polypeptide or a polynucleotide encoding the same in a biological sample. A mutation in a sequence, or the amount or biological activity of a polypeptide of the invention in a biological sample is detected.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the invention also relates to the polypeptides and / or polynucleotides of the invention in the preparation for the treatment of cancer, developmental diseases Or the use of drugs for immune diseases or other diseases caused by abnormal expression of human cytostatic protein 48.
  • 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 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 human cytostatic protein 48, causes a change in the protein to regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to human cytostatic protein 48.
  • Antagonist refers to a molecule that, when combined with human cytostatic protein 48, can block or regulate the biological or immunological activity of human cytostatic protein 48.
  • Antagonists and inhibitors can include proteins, nucleic acids, carbohydrates or any other molecule that can bind to human cytostatic protein 48.
  • Regulation refers to a change in the function of human cytostatic protein 48, including an increase or decrease in protein activity, a change in binding properties, and any other biological, functional, or immune properties of human cytostatic protein 48.
  • substantially pure ' means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify human cytostatic protein 48 using standard protein purification techniques. Basically Pure human cytostatic protein 48 can generate a single main band on a non-reducing polyacrylamide gel. The purity of human cytostatic protein 48 can be analyzed by amino acid sequence analysis.
  • 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 or Northern 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 the same or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the 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 the Clus ter method (Higgins, D. G. and P. M. Sharp (1988) Gene 73: 237-244). The Clus ter method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by:
  • the percent identity between nucleic acid sequences can also be determined by the Clus ter method or by a method known in the art such as Jotun Hein ( Hein J., (1990) Methods in emzumology 183: 625-645).
  • Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids for conservative substitutions For example, negatively charged amino acids may include Asparagus Amino acids and glutamic acid; positively charged amino acids may include lysine and arginine; amino acids with similarly hydrophilic head groups having uncharged head groups may include leucine, isoleucine and valine Amino acids; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • the "antisense strand” refers to a nucleic acid strand that is complementary to the “sense strand”.
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it.
  • This chemical dressing may be a hydrogen atom replaced by an alkyl 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? ⁇ It can specifically bind to the epitope of human cytostatic protein 48.
  • 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 cytostatic protein 48 means that human cytostatic protein 48 is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify human cytostatic protein 48 using standard protein purification techniques. Substantially pure polypeptides produce a single main band on a non-reducing polyacrylamide gel. The purity of the human cytostatic protein 48 polypeptide can be analyzed by amino acid sequence analysis.
  • the present invention provides a new polypeptide, human cell growth inhibitory protein 48, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be products that are naturally purified, or are products of chemical synthesis, or are produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques.
  • polypeptide of the invention may be glycosylated , Or can 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 cytostatic protein 48. As used herein, the terms “fragment”, “derivative” and “analog” refer to a polypeptide that substantially maintains the same biological function or activity of the human cytostatic protein 48 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 replaced with conservative or non-conservative amino acid residues, preferably conservative amino acid residues, and Substituted amino acids may or may not be encoded by the genetic code; or (II) 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); or (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) As set forth herein, such fragments, derivatives, and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a 1536 base polynucleotide sequence and its open reading frame 97-1404 encodes 435 amino acids.
  • This polypeptide has an ankyrin repeating sequence fragment domain and a characteristic sequence of the uroporphyrinogen decarboxylase protein family. It can be deduced that the human cytostatic protein 48 has an ankyrin repeating sequence fragment domain and an uroporphyrinogen decarboxylase. The structure and function represented by the protein family.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide that includes the polypeptide and a polynucleotide that includes additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be naturally occurring Allelic 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% Fi col l, 42 ° C, etc .; or (3) only between two sequences Hybridization occurs only when the identity is at least 95%, and more preferably 97%.
  • 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 cytostatic protein 48.
  • 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 cytostatic protein 48 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.
  • 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.
  • Various methods have been developed for mRNA extraction, and kits are also commercially available (Qiagene :). And the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Labora tory Manua, Coll Spring Harbor Labora tory. 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.
  • the 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 appearance or loss of marker gene function; (3) measurement Determine the transcript level of human cytostatic protein 48; (4) Detect the protein product of gene expression by immunological techniques or by measuring biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 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.
  • DM probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product of human cytostatic protein 48 gene expression.
  • ELISA enzyme-linked immunosorbent assay
  • a method using DNA technology to amplify DNA / RNA (Sa iki, et al. Sc ience 1985; 230: 1 350-1 354) 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 / RM 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 a 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 cytostatic protein 48 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology. .
  • a polynucleotide sequence encoding the human cytostatic protein 48 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 adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors expressed in bacteria (Rosenberg, et al.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain origins of replication, promoters, marker genes, and translational regulators. Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human cytostatic protein 48 and appropriate transcriptional / translational regulatory elements.
  • DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide niRNA synthesis.
  • promoters are: the l ac or trp promoter of E.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for MA expression, usually about 10 to 300 base pairs, which act on the promoter to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenovirus enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human cytostatic protein 48 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 a recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf 9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DM sequence according to the present invention or a recombinant vector containing the DNA sequence can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryote, such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human cytostatic protein 48 (Sc ience, 1984; 224: 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. 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 sequence of the human cytostatic protein 48 and ankyrin repeating sequence fragment domain and the function of the urinary porphyrinogen decarboxylase protein family of the present invention.
  • Figure 2 is a polyacrylamide gel electrophoresis image (SDS-PAGE) of the isolated human cytostatic protein 48.
  • 48I Da is the molecular weight of the protein.
  • the arrow indicates the isolated protein band. The best way to implement the present 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 RM using Quik mRNA Isolat ion Kit (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA.
  • a Smart cDNA cloning kit purchased from CI on tech) was used to insert the 00 fragment into the multiple cloning site of the pBSK (+) vector (Clontech) to transform DH5 ⁇ , and the bacteria formed a cDNA library.
  • the sequences at the 5 'and 3' ends of all clones were determined using Dye terminate cycl e react ion sequencing kit (Perkin-Elmer) and 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 0469f04 was new DNA.
  • the inserted cDNA fragments contained in this clone were determined in both directions by synthesizing a series of primers.
  • the sequence of the human cytostatic protein 48 and its encoded protein sequence of the present invention were profiled by the GCF prof le scan program (Basiclocal Alignment search tool) [Altschul, SF et al. J. Mol. Biol. 1990; 215: 403-10], performing domain analysis in databases such as Prote.
  • the human cytostatic protein 48 of the present invention is homologous to the ankyrin repeating sequence fragment domain of the domain and the uroporphyrinogen decarboxylase protein family. The homology results are shown in FIG. 1.
  • Example 3 Cloning of a gene encoding human cytostatic protein 48 by RT-PCR
  • CDNA was synthesized using fetal brain 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:
  • Primerl 5'- GCTGATGGTAAAGCTTTTGCAGAT-3 '(SEQ ID NO: 3)
  • Pr imer2 5-GCAATACAAATCTCTTTTTATTATT -3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp at the 5 ′ end of SEQ ID:
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification reaction conditions bandit containing 50 ol / L KC1 in a reaction volume of 5 0 ⁇ 1, 10 hidden ol / L Tr i s-
  • RNA extraction in one step [Ana l. 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 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1), centrifuge after mixing. The aqueous layer was aspirated, isopropanol (0.8 vol) was added and the mixture was centrifuged to obtain RM precipitate. The obtained RM precipitate was washed with 70% ethanol, dried and dissolved in water.
  • a 32P-labeled probe (approximately 2 x 10 6 cpm / ml) and RNA-transferred nitrocellulose membrane were placed in a solution at 42 ° C. C hybridization overnight, the solution contains 50% formamide-25mM KH 2 PO 4 (pH 7.4)-5 x SSC-5 x Denhardt, s solution and 200 g / ral salmon sperm DNA. After hybridization, the filters were placed in 1 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 cytostatic protein 48
  • Pr imer 3 5'- CCCCATATGATGAAAGCAGAAAACAGCCACAAT -3 '(Seq ID No: 5)
  • Pr imer4 5'- CATGGATCCCTATTCTTTACGTAGTAGCTTACA -3' (Seq ID No: 6)
  • the Ndel and BamHI restriction sites correspond to the expression vector plasmid pET 28b (+) (Novagen, Cat. No. 69865. 3) Selective endonuclease site.
  • PCR was performed using the pBS-0469f 04 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of pBS- 0469f 04 plasmid, Primer-3 and? 1 ⁇ 11161: -4 points and another!] Is 1 ( ⁇ 11101, Advantage polymerase Mix (Clontech)) 1 ⁇ 1.
  • Cycle parameters 94.C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25
  • the digestion product and plasmid pET-28 (+) were double-digested with Ndel and BamHI, respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligation products were transformed with the calcium chloride method Escherichia coli DH5 cx. Kanamycin After the LB plate (final concentration of 30 ⁇ ⁇ / ⁇ 1) was cultured overnight, positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0469f 04) with a correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method.
  • kanamycin final concentration of 3 ( ⁇ g / ml) of LB liquid medium, host strain BL21 (P ET-0469f04) incubated at 37 C to logarithmic phase, IPTG was added to a final concentration of 1 ol / Continue to culture for 5 hours. Collect the cells by centrifugation. Decompose the cells by ultrasound, collect the supernatant by centrifugation, and use an affinity chromatography column His s. Bind Quick Cartridge that can bind to 6 histidines (6His-Tag).
  • the following peptides specific for human cytostatic protein 48 were synthesized using a peptide synthesizer (product of PE): NH2-Met-Lys-Ala-Glu-Asn-Ser-His-Asn-Ala-Gly-Gln-Va l -Asp-Thr-Arg-COOH (SEQ ID NO: 7).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern blotting Trace method, Northern blotting method and copying method, etc., are all used to fix the polynucleotide sample to be tested on the filter membrane and then hybridize using basically the same steps.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer, so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthetic polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • the preferred range of probe size is 18-50 nucleotides
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements 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 which belongs to the second type of probe, is equivalent to the replacement mutation 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 membranes nitrocellulose membranes
  • Two NC membranes are required for each probe, so that they can be used in the following experimental steps.
  • the film was washed with high-strength conditions and strength conditions, respectively.
  • the 32 P-Probe (the second peak is free ⁇ - 32 P-dATP) is prepared.
  • Gene chip or gene micro matrix (DNA Mi croarray) 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 cDM are used as target DNA, including the polynucleotide of the present invention. They were respectively amplified by PCR. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and spotted on a glass with a Cartesian 7500 spotter (purchased by Cartesian Company, USA). The distance between the points on the medium is 280 ⁇ m. The spotted slides were hydrated, dried, and cross-linked in a UV cross-linker. After elution, the slides were fixed to fix the DNA on the glass slides to prepare chips. The specific method steps have been variously reported in the literature. The post-spot processing steps of this embodiment are:
  • Total mRM was extracted from normal liver and liver cancer in one step, and mRNA was purified with Oligotex mRNA Midi Kit (purchased from QiaGen).
  • Oligotex mRNA Midi Kit purchased from QiaGen.
  • the fluorescent reagent Cy3dUTP (5-Amino-propargy 1-2 'was purified by reverse transcription.
  • the probes from the above two types of tissues were hybridized with the chip in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, and washed with a washing solution (lx SSC, 0.2% SDS) at room temperature. Then use a ScanArray 3000 scanner (purchased from General Scanning, USA) to scan. The scanned image is analyzed by In gene software (Biodi scovery, USA) to calculate the Cy3 / Cy5 ratio of each point, which is less than 0. Points greater than 5 are considered genes with differential expression.
  • polypeptides of the present invention as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.
  • Histocompatibility type II protein plays an important regulatory role in the development of the organism and the immune system. The lack of reaction factors necessary for the gene expression of histocompatibility type II protein will lead to the loss of histocompatibility type II protein. It is usually associated with the development of some developmental disorders and autosomal immunodeficiency diseases.
  • RFXANK is the third subunit of the expression regulator protein (RFX) of histocompatibility type II protein.
  • RFXAM is also an ankyrin-like protein. Mutations in the ankyrin repeat sequence domain in its sequence will directly cause the abnormal function of the protein. Proteins containing this domain are usually associated with some common hereditary hemolytic anemia in the body. happen closely related.
  • the polypeptide of the present invention is a polypeptide containing an ankyrin repeating sequence fragment domain of the expression regulatory factor subunit 3 RFXANK of a compatible class II protein, and has characteristic sequence fragments of the uroporphyrinogen decarboxylase protein family.
  • Urinary porphyrinogen decarboxylase is necessary for the heme biosynthetic pathway. It is highly expressed in tissues related to blood cell metabolism and participates in the production of blood cells in related tissues. The loss of uroporphyrinogen decarboxylase is usually closely related to the occurrence of some family genetic diseases in humans, such as: fPCT and hepatic hematopoiesis.
  • the present invention is a human cell growth inhibiting abnormal protein expression 48 will produce a variety of human diseases, in particular major histocompatibility antigen associated diseases, autoimmune diseases, blood diseases, disorders embryonic development, growth Obstructive diseases, including but not limited to:
  • Human major histocompatibility antigen-related diseases blood group incompatibility transfusion reactions, neonatal hemolytic anemia, rheumatoid arthritis, chronic active hepatitis, primary dry syndrome, acute anterior uveitis, gonococcal infection Post-arthritis, ankylosing spondylitis, hemochromatosis, immune complex glomerulonephritis, myocarditis after gonococcal infection
  • Autoimmune diseases systemic lupus erythematosus, scleroderma, polymyositis, xerostomia syndrome, nodular polyarteritis, Wegener's granulomatosis, myasthenia gravis, Guillain-Barre syndrome, autoimmune Hemolytic anemia, immune thrombocytopenic purpura, granulocytopenia, autoimmune interstitial nephritis, autoimmune gastritis, insulin autoimmune syndrome, autoimmune thyroid disease, autoimmune heart disease
  • Hematological diseases hereditary hemolytic anemia, purpura, various anemias such as thalassemia, megaloblastic anemia, malignant anemia, aplastic anemia, hemolytic anemia, bone marrow anemia, anemia secondary to chronic diseases, Polycythemia, hereditary oval red blood cells, acute leukemia, chronic myelogenous leukemia, Chronic lymphocytic leukemia, chronic monocytic leukemia, lymphoid reticulum, malignant lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma
  • Fetal developmental disorders congenital abortion, cleft palate, limb loss, limb differentiation disorder, cryptorchidism, atrial septal defect, neural tube defect, congenital hydrocephalus, iris defect, congenital glaucoma or cataract, congenital deafness
  • Abnormal expression of the human cytostatic protein 48 of the present invention will also generate certain tumors and the like.
  • the polypeptide of the present invention can be directly used in the treatment of diseases, for example, it can treat various diseases, especially diseases related to the major histocompatibility of human body, autoimmune diseases, and hematological diseases. , Embryonic disorders, growth disorders, certain tumors, etc.
  • the invention also provides methods of screening compounds to identify agents that increase (agonist) or suppress (antagonist) human cell growth inhibitory protein 48.
  • Agonists enhance human cytostatic protein 48 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • a mammalian cell or a membrane preparation expressing human cytostatic protein 48 can be cultured together with a labeled human cytostatic protein 48 in the presence of a drug. The ability of the drug to increase or suppress this interaction is then determined.
  • Antagonists of human cytostatic protein 48 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human cytostatic protein 48 can bind to human cytostatic protein 48 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions.
  • human cytostatic protein 48 When screening compounds as antagonists, human cytostatic protein 48 can be added to bioanalytical assays to determine whether the compound is an antagonist by measuring the effect of the compound on the interaction between human cytostatic protein 48 and its receptor. . 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 cytostatic protein 48 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, generally 48 molecules of human cytostatic protein should be labeled.
  • the present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against the human cytostatic protein 48 epitope. These antibodies include (but are not limited to): Polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human cytostatic protein 48 directly into 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. Wait. Techniques for preparing monoclonal antibodies to human cytostatic protein 48 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma Technology, EBV-hybridoma technology, etc. Chimeric antibodies that bind human constant regions and 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 cell growth inhibitory protein 48.
  • Antibodies against human cytostatic protein 48 can be used in immunohistochemical techniques to detect human cytostatic protein 48 in biopsy specimens.
  • Monoclonal antibodies that bind to human cytostatic protein 48 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 cytostatic protein 48 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 disulfide exchange.
  • This hybrid antibody can be used to kill human cytostatic 48-positive cells .
  • the antibodies of the present invention can be used to treat or prevent diseases related to human cytostatic protein 48. Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human cytostatic protein 48.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human cytostatic protein 48 levels.
  • tests are well known in the art and include FI SH assays and radioimmunoassays.
  • the level of human cytostatic protein 48 detected in the test can be used to explain the importance of human cytostatic protein 48 in various diseases and to diagnose diseases in which human cytostatic protein 48 plays a role.
  • the polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzyme, and can be analyzed by one-dimensional or two-dimensional or three-dimensional gel electrophoresis, and more preferably by mass spectrometry encoding
  • the polynucleotide of human cytostatic protein 48 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 cytostatic protein 48.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed for Expression of mutant human cytostatic protein 48 to inhibit endogenous human cytostatic protein 48 activity.
  • a variant human cytostatic protein 48 may be a shortened human cytostatic protein 48 that lacks a signal transduction domain. Although it can bind to downstream substrates, it lacks signal transduction activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of human cytostatic protein 48.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus and the like can be used to transfer a polynucleotide encoding human cell growth inhibitory protein 48 into cells.
  • recombinant viral vectors carrying a polynucleotide encoding human cytostatic protein 48 can be found in the literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human cytostatic protein 48 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 DNA
  • ribozymes that inhibit human cell growth inhibitory protein 48 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 A for endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as solid-phase phosphoramidite chemical synthesis, which is 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 vector's RNA polymerase promoter.
  • 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 cytostatic protein 48 can be used for the diagnosis of diseases related to human cytostatic protein 48.
  • the polynucleotide encoding human cytostatic protein 48 can be used to detect the expression of human cytostatic protein 48 or the abnormal expression of human cytostatic protein 48 in a disease state.
  • a DNA sequence encoding human cytostatic protein 48 can be used to hybridize biopsy specimens to determine the expression of human cytostatic protein 48.
  • 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 micro array or a DNA chip (also called a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Human cytokine 48 specific primers can be used for RNA-polymerase chain reaction (RT-PCR) amplification in vitro to detect the transcription of human cytokine 48 Detection of mutations in the human cytostatic protein 48 gene can also be used to diagnose human cytostatic protein 48-related diseases.
  • Human cytostatic protein 48 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human cytostatic protein 48 DNA sequence.
  • Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • 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 pre-selection of hybridization to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendelian Inheritance in Man (available online with Johns Hopkins University Welch Medica l Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the differences in cDNA or genomic sequences between the affected and unaffected individuals need to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable using cDNA sequence-based PCR. Based on the resolution capabilities of current physical mapping and gene mapping technologies, The cDNA of the disease-related chromosomal region can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution and one gene per 20 kb).
  • 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 cytostatic protein 48 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human cytostatic protein 48 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 48 d'inhibition de la croissance cellulaire, 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 48 d'inhibition de la croissance cellulaire.
PCT/CN2001/000076 2000-01-28 2001-01-21 Nouveau polypeptide, proteine humaine 48 d'inhibition de la croissance cellulaire, et polynucleotide codant pour ce polypeptide Ceased WO2001055379A1 (fr)

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AU31490/01A AU3149001A (en) 2000-01-28 2001-01-21 A novel polypeptide-human cell growth inhibition protein 48 and the polynucleotide encoding said polypeptide

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CN 00111614 CN1307056A (zh) 2000-01-28 2000-01-28 一种新的多肽——人细胞生长抑制蛋白48和编码这种多肽的多核苷酸
CN00111614.2 2000-01-28

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] 11 November 1999 (1999-11-11), NAGASE T. ET AL., accession no. NCBI Database accession no. BAA86537.1 *
DATABASE GENBANK [online] 29 October 1999 (1999-10-29), CUMMINGS P., accession no. NCBI Database accession no. T24158 *

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