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WO2002012315A1 - Nouveau polypeptide, proteine mid1 10.56 du gene de type « ring finger », et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, proteine mid1 10.56 du gene de type « ring finger », et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002012315A1
WO2002012315A1 PCT/CN2001/001047 CN0101047W WO0212315A1 WO 2002012315 A1 WO2002012315 A1 WO 2002012315A1 CN 0101047 W CN0101047 W CN 0101047W WO 0212315 A1 WO0212315 A1 WO 0212315A1
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Prior art keywords
polypeptide
polynucleotide
ring finger
protein
finger gene
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc
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Shanghai Biowindow Gene Development Inc
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Priority to AU14904/02A priority Critical patent/AU1490402A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • a new poly-ring refers to a certain MIDI protein 10. 56 and a polynucleotide encoding the polypeptide TECHNICAL FIELD
  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide ⁇ ⁇ refers to a gene MIDI protein 10.56, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a method and application for preparing such polynucleotides and polypeptides. technical background
  • the terminus tends to form a terminus deletion, and the consequence is a dominant or recessive growth syndrome on the X chromosome (Bal labio and Andia, 1992).
  • Genealogy analysis of some related patients found a series of disease-causing regions located on chromosome Xp22 and many related pathogenic genes, such as X-chromosome-related eye albinism genes (0A1; Bassi et al., 1995), Ka lman syndrome (KALI; Franco et al., 1991), enamel hypoplasia (Amelogenin, AMBLX; Lagers trom et al., 1991) and so on.
  • Small eye and linear skin defect disease (MLS) is an X-chromosomal dominant male lethal genetic development disorder.
  • MIDI is one of them. It is a ring finger gene and a gene that causes the X chromosome-related Opitz G syndrome (Quader i et al. , 1997).
  • MIDI clone of MIDI was obtained from a human infant kidney cDNA library, containing an open reading frame of 2001Bp, terminated by a TGA stop codon within the frame.
  • the cDNA sequence is the same as a RING loop finger gene subtype (Freemont et al., 1991), which contains a RING loop finger region, two B-box structures, and a coiled coil region. These regions are relatively concentrated on half of the sequence near the 5 'end. In addition, there is an additional conserved region at the tail of the C-terminus.
  • the structurally conserved ring finger region and two B-boxes are encoded by the first exon 1 containing 716Bp, the coiled-coil region is encoded by exons 2 and 3, and the C-terminal region is encoded by exons Sub 8 and 9 co-encoded
  • the symptoms of MLS patients are mainly deformed eyes (such as small eyes, congenital fissure of the eye, corneal opacity), abnormalities of the central nervous system (such as corpus callosum hypoplasia), and linear plaques on the neck and face. Because they are male-killing, all known patients are female.
  • MIDI gene 5 changes in untranslated regions will cause specific imbalances that can alter growth or tissue-specific gene expression. Mutations in the coding sequence of the MIDI gene will result in a significant non-male-killing phenotype, the X chromosome-related Opitz G syndrome (Quader i et al., 1997).
  • the ring finger gene MIDI protein 10.56 protein plays an important role in regulating important functions of the body such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, so there has been a need to identify more participation in the field
  • the ring finger of these processes is the MIDI protein 10.56 protein, especially the amino acid sequence of this protein is identified.
  • the new ring finger gene MIDI protein 10. 56 The isolation of the protein-coding gene also provides the basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for the disease, so isolation of its coding DNA is very important.
  • 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 a ring finger gene MIDI protein 10.56.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a ring finger gene MIDI protein 10.56.
  • Another object of the present invention is to provide a method for producing a ring finger gene MIDI protein 10.56.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention, the ring finger gene MIDI protein 10.56.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention, the ring finger gene MIDI protein 10.56.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to the abnormality of the ring finger gene MIDI protein 10.56. Summary of invention
  • 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 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 1404-1694 in SEQ ID NO: 1; and (b) a sequence having 1-3119 in SEQ ID NO: 1 Sequence of bits.
  • the present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of the ring finger gene MIDI protein 10.56 protein, which comprises utilizing the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for in vitro detection of a disease or susceptibility to disease associated with abnormal expression of the ring finger gene MIDI protein 10.56 protein, comprising detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, or Detection of 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 treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of the ring finger gene MIDI protein 10.56.
  • FIG. 1 is a comparison diagram of gene chip expression profiles of the ring finger gene MIDI protein 10.56 and the ring finger gene MIDI protein of the present invention.
  • the upper figure is a graph of the expression profile of the ring finger gene MIDI protein 10. 56, and the lower figure is the graph of the expression profile of the ring finger gene MIDI protein.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated ring finger gene MIDI protein 10.56.
  • llkDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Nucleic acid sequence refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and may also refer to the genome or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a “variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.
  • An "agonist” is a molecule that, when combined with the ring-finger gene MIDI protein 10.56, 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 the ring finger gene MIDI protein 10.56.
  • Antagonist or “inhibitor” means that when combined with the ring finger gene MIDI protein 10.56, a A molecule that can block or regulate the biological activity or immunological activity of the ring finger gene MIDI protein 10.56.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that can bind the ring finger gene MIDI protein 10.56.
  • Regular refers to a change in the function of the ring finger gene MIDI protein 10.56, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological, functional or immune properties of the ring finger gene MIDI protein 10.56. Change.
  • substantially pure means substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated.
  • Those skilled in the art can purify the ring finger gene MIDI protein 10.56 using standard protein purification techniques.
  • the substantially pure ring finger gene MIDI protein 10.56 produces a single main band on a non-reducing polyacrylamide gel.
  • the ring finger gene MIDI protein 10. 56 The purity of the 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. "Partially homologous,” refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. The inhibition of such hybridization can be achieved by hybridization under conditions of reduced stringency (Southern blotting) Or Northern blot, etc.) to detect. Basically 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 level of stringency is reduced. The conditions allow non-specific binding because conditions with reduced stringency require that the two sequences bind to each other as a specific or selective interaction.
  • 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 the Clus ter method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). 0 The Clus ter method groups each group by checking the distance between all pairs. The sequences are arranged in 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:
  • the assay may be Jotun Hein percent identity between nucleic acid sequences Clus ter or a method well known in the art (Hein J., (1990) Methods in enzymology 183: 625-645) 0 "Similarity” refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to HFP or a chemical modification of its nucleic acid. This chemical modification may be a substitution of a hydrogen atom with a fluorenyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and? ⁇ It can specifically bind to the epitope of the ring finger gene MIDI protein 10. 56.
  • 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 ring finger gene MIDI protein 10. 56 refers to the ring finger gene MIDI protein 10. 56 and is substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify the ring finger gene MIDI protein 10.56 using standard protein purification techniques. Essentially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. The ring finger gene MIDI protein 10. 56 The purity of the peptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, the ring finger gene MIDI protein 10.56, 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 polypeptide of the present invention may be a naturally purified product or a chemically synthesized product Products, or produced using recombinant technology from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of the ring finger gene MIDI protein 10.56.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity as the ring finger gene MIDI protein 10.56 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 ( ⁇ ) such a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or (in) such One, 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); or (IV) such a polypeptide sequence in which the additional amino acid sequence is fused into the mature polypeptide ( Such as leader sequences or secreted sequences or sequences used to purify this polypeptide or protease sequences).
  • 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 polynucleotide sequence of 3119 bases in length and its open reading frame 1404-1694 encodes 96 amino acids.
  • this peptide has a similar expression profile to the ring finger gene MIDI protein, and it can be inferred that the ring finger gene MIDI protein 10. 56 has a similar function as the ring finger gene MIDI protein.
  • the polynucleotide of the present invention may be in the form of DM 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 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.
  • the term "polynucleotide encoding a polypeptide" is meant to include polynucleotides that encode such polypeptides and polynucleotides that include additional coding and / or noncoding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide 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 (with at least 501 ⁇ 2, 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, 6 (TC; or (2) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficol 1, 42 'C, etc .; or (3) only between the two sequences Hybridization occurs only when the identity is at least 95%, and more preferably 97%.
  • the 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 the ring finger gene MIDI protein 10.56.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence of the loop finger gene MIDI protein 10.56 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DM sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating cDNA of interest is to isolate mRM from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • 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) DM-DM or DNA-RM hybridization; (2) the appearance or loss of marker gene function; (3) the level of the transcript of the ring finger gene MIDI protein 10. 56; (4) Detecting the protein product of gene expression by immunological technology or measuring biological activity. The above methods can be used alone or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably 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).
  • 56 gene expression protein product 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 using DNA technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein Select and synthesize using conventional methods.
  • the amplified DM / RNA fragment 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 measured 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, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using the ring finger gene MIDI protein 10.56 coding sequence, and the recombinant technology to produce the present invention Polypeptide method.
  • the polynucleotide sequence encoding the loop finger gene MIDI protein 10.56 can be inserted into a vector to form a recombinant vector containing the polynucleotide of the present invention.
  • carrier refers to the art Of bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors.
  • 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.
  • pMSXND expression vectors expressed in mammalian cells Lee and Na thans, J Bio Chera. 263: 3521, 1988
  • baculovirus-derived vectors expressed in insect cells in short, as long as it can be replicated and stabilized in a host, any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequence encoding the ring finger gene MIDI protein 10.56 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DM synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manual, Cold Spin Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation, 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 expressed by DM, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Examples include 100 to 270 base pair SV40 enhancers on the late side of the replication initiation point, polyoma enhancers and adenovirus enhancers on the late side of the replication initiation point.
  • 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 a loop finger gene MIDI protein 10.56 or a recombinant vector containing the polynucleotide can be transformed or transferred into a host cell to form a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • 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 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 eukaryote, the following DM transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce a recombinant ring finger gene MIDI protein 10. 56 (Science, 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. If necessary, the recombinant protein can be isolated and purified by various separation methods using its 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
  • 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.
  • the ring finger gene MIDI protein is a protein associated with small eyes and linear skin defects (MLS).
  • MLS patients are mainly deformed eyes (such as small eyes, congenital fissure of the eye, corneal opacity), abnormalities of the central nervous system (such as hypoplasia of the corpus callosum), and linear plaques on the neck and face. Therefore, it is believed that the ring finger gene MIDI protein is related to some facial, facial features, and embryonic developmental abnormalities of the nervous system. Abnormal expression in the body can cause disorders in the embryonic development of related systems, which can lead to related diseases happened.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of the human ring finger gene MIDI protein, and both have similar biological functions.
  • the polypeptide of the present invention is related to embryo developmental abnormalities of some eyes and nervous system in vivo, and its abnormal expression can cause disorders of embryonic development of related systems, and then lead to the occurrence of related diseases. These diseases include, but are not limited to:
  • neural tube defects no cerebellar malformation, spina bifida, spinal meningocele, hydrocephalous meningocele), hydrocephalus in / outside the brain, etc .
  • iris defect iris defect
  • pupil retention congenital cataract
  • congenital glaucoma solo / no / small eye deformity
  • congenital deafness congenital deafness
  • auricle deformity cleft lip, palate, cleft palate, cervix, neck Fistula, etc.
  • polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used for the treatment of various diseases, such as facial, facial features, and embryonic developmental disorders of the nervous system, small eyes and linear skin defects. (MLS), etc.
  • diseases such as facial, facial features, and embryonic developmental disorders of the nervous system, small eyes and linear skin defects. (MLS), etc.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) the ring finger gene MIDI protein 10.56.
  • Agonists enhance the ring finger gene MIDI protein 10.56 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 the ring finger gene MIDI protein 10. 56 can be cultured with a labeled ring finger gene MIDI protein 10. 56 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of the ring finger gene MIDI protein 10.56 include antibodies, compounds, receptor deletions, and the like that have been screened.
  • the antagonist of the ring finger gene MIDI protein 10.56 can bind to the ring finger gene MIDI protein 10.56 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 function. biological functions.
  • the ring finger gene MIDI protein 10.56 can be added to the bioanalytical assay, and the compound can be determined by measuring the effect of the compound on the interaction between the ring finger gene MIDI protein 10. 56 and its receptor. Whether it is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Peptide molecules capable of binding to the ring finger gene MIDI protein 10.56 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. During the screening, the ring finger gene MIDI protein 10.56 molecules should generally be labeled.
  • the present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides an antibody against the ring finger gene MIDI protein 10. 56 epitope.
  • These antibodies include (but are not limited to In): Polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by ring finger gene MIDI protein 10.56 by direct injection in immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • adjuvants can be used to enhance the immune response, including but not limited to Freund's Adjuvant, etc.
  • Techniques for preparing monoclonal antibodies of the ring finger gene MIDI protein 10.56 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human beta cell hybridoma Technology, EBV-hybridoma technology, etc.
  • An inlay antibody combining a human constant region and a non-human variable region can be produced using existing technologies (Morri son 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 the MIDI protein 10.56.
  • Antibodies to the ring finger gene MIDI protein 10. 56 can be used in immunohistochemical techniques to detect the ring finger gene MIDI protein 10. 56 in biopsy specimens.
  • Monoclonal antibodies that bind to the ring finger gene MIDI protein 10.56 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. Such as ring finger gene MIDI protein 10.
  • 56 High affinity monoclonal antibodies can covalently bind to bacterial or phytotoxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a sulfhydryl cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill the ring finger gene MIDI protein 10. 56 positive Cell.
  • the antibodies of the present invention can be used to treat or prevent diseases related to the ring finger gene MIDI protein 10.56.
  • the proper dose of antibody can stimulate or block the production or activity of the ring finger gene MIDI protein 10.56.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of the ring finger gene MIDI protein 10.56.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of the ring finger gene MIDI protein 10.56 detected in the test can be used to explain the importance of the ring finger gene MIDI protein 10. 56 in various diseases and to diagnose the role of the ring finger gene MIDI protein 10. 56. disease.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • the polynucleotide encoding the ring finger gene MIDI protein 10.56 can also be used for a variety of therapeutic purposes.
  • Gene Therapeutic techniques can be used to treat abnormal cell proliferation, development, or metabolism caused by the non-expression or abnormal / inactive expression of the ring finger gene MIDI protein 10.56.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated ring finger gene MIDI protein 10.56 to inhibit endogenous ring finger gene MIDI protein 10.56 activity.
  • a variant ring finger gene MIDI protein 10.56 may be a shortened ring finger gene MIDI protein 10.56, although it can bind to downstream substrates, but lacks signal transduction activity.
  • the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of the ring finger gene MIDI protein 10.56.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding the ring finger gene MIDI protein 10.56 into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a loop finger gene MIDI protein 10.56 can be found in existing literature (Sambrook, et al.).
  • a polynucleotide encoding the MIDI protein 10.56 of the recombinant ring finger gene can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: injecting the polynucleotide directly into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit the ring finger gene MIDI protein 10.56 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained by any RNA or DNA synthesis technology. For example, solid-phase phosphoramidite chemical synthesis to synthesize oligonucleotides has been 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. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding the ring finger gene MIDI protein 10. 56 can be used for the diagnosis of diseases related to the ring finger gene MIDI protein 10. 56.
  • the polynucleotide encoding the ring finger gene MIDI protein 10.56 can be used to detect the expression of the ring finger gene MIDI protein 10. 56 or the abnormal expression of the ring finger gene MIDI protein 10. 56 in a disease state.
  • the DNA sequence encoding the ring finger gene MIDI protein 10. 56 can be used to hybridize biopsy specimens to determine the expression status of the ring finger gene MIDI protein 10. 56.
  • Hybridization techniques include Southern blotting, Northern blotting, and in situ hybridization.
  • RNA-polymerase chain reaction RT-PCR
  • Detection of ring finger gene MIDI protein 10.56 mutations can also be used to diagnose ring finger gene MIDI protein 10.56-related diseases.
  • the ring finger gene MIDI protein 10.56 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild type ring finger gene MIDI protein 10.56 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, Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DM sequences on a chromosome.
  • a PCR primer (preferably 15-35bp) is prepared from the cDNA, and the sequence can be located on the chromosome. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize 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 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 individual, The mutation may be the cause of the disease. Comparing diseased and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable with cDM 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.
  • the ring finger gene MIDI protein 10. 56 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dose range of the ring finger gene MIDI protein 10.56 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. Examples
  • Total human fetal brain RNA 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 was reverse transcribed to form cDNA.
  • the Smart cDNA cloning kit (purchased from C1 on tech) was used to insert the 00 fragment into the multiple cloning site of the pBSK (+) vector (Clontech) to transform DH5a.
  • the bacteria formed a CDM library.
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 An automatic sequencer (Perkin-Elmer) determined the sequences at the 5 'and 3' ends of all clones. The determined cDNA sequence was compared with an existing public D sequence database (Genebank), and it was found that the cDNA sequence of one of the clones, 0068 gl 0, was new DNA. A series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • the 0068gl0 clone contains a full-length cDNA of 3119bp (as shown in Seq ID N0: l), and has a 290bp open reading frame (0RF) from 1404bp to 1694bp, encoding a new protein (such as Seq ID NO : Shown in 2).
  • This clone pBS-0068gl0 and the encoded protein was named the ring finger gene MIDI protein 10.56.
  • Example 2 The gene encoding the ring finger gene MIDI protein 10.56 was cloned by the RT-PCR method. Total fetal brain cells were used as a template, and ol igo-dT was used as a primer for reverse transcription reaction to synthesize cDNA. , Using the following primers for PCR amplification:
  • Primerl 5,-AACCCTCACTAAAGGGAACAAAAG -3 '(SEQ ID NO: 3)
  • Primer2 5,-AATGTCTCATTTATTGAGAATATT -3 '(SEQ ID NO: 4)
  • Pr imerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Primer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.
  • Conditions for the amplification reaction 50 mmol / L KCl, 10 mfflol / L Tr is-HCl pH 8.5, 1. 5 ramol / L MgCl 2 , 200 ⁇ 1 / ⁇ dNTP, l Opmol primer, 1U in a 50 ⁇ 1 reaction volume Taq DNA polymerase (Clontech).
  • the 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 technicallyAt the same time, set ⁇ -act in as positive during RT-PCR Controls and template blanks were negative controls.
  • the amplified products were purified using a QIAGEN kit and linked to a pCR vector (Invitrogen) using a TA cloning kit.
  • DNA sequence analysis results showed that the DNA sequence of the PCR product was in accordance with SEQ ID NO : The l-3119bp shown in Fig. 1 are exactly the same.
  • Example 3 Northern blot analysis of MIDI protein 10.56 gene expression The total MA was extracted in one step [Anal. Biochem 1987, 162, 156-159], this method Including acid guanidinium thiocyanate phenol-chloroform extraction.
  • the tissue was homogenized with 4M guanidine isothiocyanate-25raM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1 / 5 volume of chloroform-isoamyl alcohol (49: 1), mixed and centrifuged. Aspirate the aqueous phase layer, add isopropanol (0.8 volume) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA precipitate was used 70 ° /. Wash with ethanol, dry and dissolve in water. Use 20 ⁇ 2 RNA, containing 2 Electrophoresis was performed on a 1.2 % agarose gel of 0mM 3- (N-? 1 * generation) propanesulfonic acid (pH7.
  • the 32P -labeled DNA probe was prepared by a random primer method using ⁇ - 32P dATP.
  • the DNA probe used was the PCR amplified loop finger gene MIDI protein 10.56 coding region shown in Figure 1 Sequence (1404bp to 1694bp).
  • a 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RM was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 (pH7.
  • Primer3 5'-CATGCTAGCATGGACCTTCTGTATAAAAACCCC-3 '(Seq ID No: 5)
  • Primer4 5'-CCCGAATTCCTAGCATCTAGGAAACAAAGTGAG-3' (Seq ID No: 6)
  • the 5 'ends of these two primers contain Nhel and EcoRI restriction sites, respectively.
  • the coding sequences of the 5 'and 3' ends of the target gene are followed, respectively.
  • the Nhel and EcoRI restriction sites correspond to the selection on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865. 3). Sex endonuclease site.
  • PCR reaction conditions were: 1 in a total volume of 50 ⁇ plasmid pBS- 0068gl0 containing 10pg, primer Primer- 3 and Primer- 4 are 10pmol, Advantage polymerase Mix (Clontech Products) 1 ⁇ 1. Cycle parameters: 94 ° C 20s, 60. C 30s, 68 ° C 2 min, a total of 25 cycles. Nhel and EcoRI were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligated product was transformed into coliform bacteria DH5a by the calcium chloride method, and cultured overnight in LB plates containing kanamycin (final concentration 30 ⁇ ⁇ / ⁇ 1). Positive clones were selected by colony PCR method and sequenced. A positive clone (PBT-0068 g 10) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method.
  • the host bacteria BL21 (pET-0068glO) was cultured at 37 ° C to the logarithmic growth phase, IPTG was added to the final concentration limnol / L, and continued Incubate for 5 hours. The bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation and chromatographed using an affinity column His. Bind Quick Cartridge (product of Novagen) capable of binding to 6 histidines (6His-Tag). The purified target protein ring finger gene MIDI protein 10.56.
  • a peptide synthesizer (product of PE company) was used to synthesize a peptide specific to the following MIDI protein 10.56: NH2-Met-Asp-Leu-Leu-Tyr-Lys-Asn-Pro-Leu-Ala-Phe-Phe -Phe-Thr-Ser-C00H (SEQ ID NO: 7).
  • the polypeptide is coupled with 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 using a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern imprinting, Northern blotting, and copying methods. They all use the same steps to immobilize the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature), so that the hybridization background is reduced and only strong specific signals are retained.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention as hybridization probes should follow the following principles and several aspects to be considered: 1.
  • 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, then 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 membrane nitrocellulose membrane
  • the 32 P-Probe (the second peak is free ⁇ - 32 P-dATP) is prepared.
  • the sample membrane was placed in a plastic bag, and 3-10 mg of prehybridization solution (lOxDenhardt-s; 6xSSC, 0.1 mg / ml CT DM (calf thymus DNA)) was added. After sealing the mouth of the bag, shake at 68 ° C for 2 hours.
  • prehybridization solution lOxDenhardt-s; 6xSSC, 0.1 mg / ml CT DM (calf thymus DNA)
  • Gene microarrays or DNA microarrays are new technologies currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • the specific method steps have been reported in the literature. For example, see DeRi si, JL, Lyer, V. & Brown, PO (1997) Science 278, 680-686. And Hel le, RA, Schema, M. , Chai, A., Shalom, D., (1997) PNAS 94: 2150-2155.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were respectively amplified by PCR, and the concentration of the amplified product was adjusted to about 500 ng / ul after purification.
  • the Cartesian 7500 spotter (purchased from Cartesian Company, USA) was spotted on the glass medium, between the spots The distance is 280 ⁇ m.
  • the spotted slides were hydrated and dried, cross-linked in a UV cross-linker, and dried after elution to fix the DM on the glass slide to prepare chips.
  • the specific method steps have been reported in the literature.
  • the sample post-processing steps in this embodiment are:
  • the probes from the two types of tissues and the chip were hybridized in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, and then washed with a washing solution (1 ⁇ SSC, 0.2 SDS) at room temperature. Scanning was performed with a ScanArray 3000 scanner (purchased from General Scanning, USA), and the scanned images were analyzed and processed with Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are fetal brain, bladder mucosa, PMA + Ecv304 cell line, LPS + Ecv304 cell line, thymus, normal fibroblasts 1024NC, Fibroblast, growth factor stimulation, 1024NT, scar formation fc growth factor stimulation, 1013HT, 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. Based on these 18 Cy3 / Cy5 ratios, a bar graph is drawn ( Figure 1). It can be seen from the figure that the ring finger gene MIDI protein 10.56 and the ring finger gene MIDI protein expression profiles according to the present invention are very similar.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine MID1 10.56 du gène de type « RING finger », 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 malformations apparaissant lors du développement de l'embryon affectant le visage, les cinq organes et le système nerveux et de microphtalmie avec déficiences cutanées linéaires. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant la protéine MID1 10.56 du gène de type « RING finger ».
PCT/CN2001/001047 2000-06-26 2001-06-25 Nouveau polypeptide, proteine mid1 10.56 du gene de type « ring finger », et polynucleotide codant ce polypeptide Ceased WO2002012315A1 (fr)

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CN 00116744 CN1331086A (zh) 2000-06-26 2000-06-26 一种新的多肽——环指基因mid1蛋白10.56和编码这种多肽的多核苷酸

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Title
GENOMICS, vol. 51, no. 2, 1998, pages 251 - 261 *
GENOMICS, vol. 62, no. 3, 1999, pages 385 - 394 *
PROC. NATL. ACAD. SCI. USA, vol. 96, no. 6, 1999, pages 2794 - 2799 *

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