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WO2002026813A1 - Nouveau polypeptide, facteur humain 222.88 associe a la proliferation de myocytes, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, facteur humain 222.88 associe a la proliferation de myocytes, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002026813A1
WO2002026813A1 PCT/CN2001/000912 CN0100912W WO0226813A1 WO 2002026813 A1 WO2002026813 A1 WO 2002026813A1 CN 0100912 W CN0100912 W CN 0100912W WO 0226813 A1 WO0226813 A1 WO 0226813A1
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Prior art keywords
polypeptide
polynucleotide
muscle cell
human muscle
binding factor
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PCT/CN2001/000912
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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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Application filed by Shanghai Biowindow Gene Development Inc. filed Critical Shanghai Biowindow Gene Development Inc.
Priority to AU2001289518A priority Critical patent/AU2001289518A1/en
Publication of WO2002026813A1 publication Critical patent/WO2002026813A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide ⁇ ⁇ muscle cell enhanced binding factor 222. 88, and a polynucleotide sequence encoding the polypeptide. The invention also relates to methods and applications for preparing such polynucleotides and polypeptides.
  • Muscle cell enhancement binding factor 2 site is an important role of protein binding to a variety of muscle-specific enhancement factors and promoters. Muscle-enhancing factors bind to the nucleus of muscle and brain cells in the body to regulate the development and growth of relevant tissues in the body.
  • Myocyte-enhanced binding factor 2 has been formed into an independent protein family in the body-a myocyte-enhanced binding factor 2 transcription factor protein family. Members of this protein family are highly expressed in skeletal muscle and brain. Later stimulated expression [Jolm C. Mcdermot t, M. Cr ist ina Cardoso et a l., 1993, Molecular and Ce l lular Biology, 13 (4): 2564-2577]
  • myocyte-enhanced binding factor 2 is highly expressed in the muscle differentiation and brain tissues of the organism, and this protein binds to the corresponding myocyte-enhanced factor in vivo to synergistically regulate the growth and development of muscle tissue and nervous system.
  • the protein is highly expressed in skeletal muscle and brain, and it regulates muscle formation and nervous system development in vivo.
  • the mutation or abnormal expression of this protein will lead to abnormal development of the corresponding tissues and cells in the organism. It is usually associated with a variety of disorders related to the development of nervous system in the organism, abnormal development of muscle tissue and tumors and cancers of related tissues. happen closely related.
  • the protein can also be used for the diagnosis and treatment of various related diseases mentioned above.
  • the human muscle cell enhanced binding factor 222.88 protein plays an important role in regulating important functions of the body such as cell division and embryo development, and it is believed that a large number of proteins are involved in these regulatory processes, so more needs to be identified in the art
  • the human muscle cell enhanced binding factor 222.88 protein involved in these processes, in particular the amino acid sequence of this protein is identified.
  • New human muscle cell enhanced binding factor 222. 88 The isolation of the protein-coding gene also provides a basis for research to determine the role of the protein in health and disease states. This protein may form the basis for developing diagnostic and / or therapeutic drugs for the disease, so it is important to isolate its code for DM. Object of the 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 muscle cell enhanced binding factor 222.88.
  • Another object of the present invention is to provide a genetically engineered host cell comprising a polynucleotide encoding human muscle cell enhanced binding factor 222.88.
  • Another object of the present invention is to provide a method for producing human muscle cell enhanced binding factor 222. 88.
  • Another object of the present invention is to provide antibodies against the polypeptide of the present invention-human muscle cell enhanced binding factor 222. 88.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors against the polypeptide of the present invention, human muscle cell enhanced binding factor 222.88.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to the abnormality of human muscle cell enhanced binding factor 222.88. 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 425-1051 in SEQ ID NO: 1; and (b) a sequence having 1-1367 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 human muscle cell enhanced binding factor 222.88 protein activity, 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 detecting a disease or disease susceptibility related to abnormal expression of human muscle cell enhanced binding factor 222.88 protein in vitro, comprising detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, Alternatively, 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 present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention in the manufacture of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of human muscle cell enhanced binding factor 222.88.
  • FIG. 1 is a myocyte enhancer factor binding 222.88 and human myocyte enhancer binding factor gene chip expression profile comparison of FIG.
  • the upper graph is a graph of the expression profile of human muscle cell enhanced binding factor 222.88
  • the lower graph is the graph of the expression profile of human muscle cell enhanced binding factor 2.
  • 1-bladder mucosa 2-PMA + EcWO 4 Cell line, 3-LPS + Ecv304 cell line thymus, 4-normal fibroblasts 1024NC, 5-Fibroblas t, growth factor stimulation, 1024NT, 6-scarf to fc growth factor stimulation, 1013HT, 7-scar to fc unused growth Factor stimulation, 1013HC, 8-bladder cancer construct cell EJ, 9-bladder cancer, 10-bladder cancer, 11-liver cancer, 12-liver cancer cell line, 13-fetal skin, 14-spleen, 15-prostate cancer, 16 -Jejunum adenocarcinoma, 17 cardia cancer.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of isolated human muscle cell enhanced binding factor 222.88. 23 kDa is the molecular weight of the protein. The arrow indicates the isolated protein band.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a “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” refers to a molecule that, when combined with human muscle cell enhanced binding factor 222.88, can cause the protein to change, thereby regulating the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to human muscle cell enhanced binding factor 222.88.
  • Antagonist or “inhibitor” means that when combined with human muscle cell enhanced binding factor 222.88, A molecule that can block or regulate human muscle cells to enhance the biological or immunological activity of binding factor 222.88.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind to human muscle cell enhanced binding factor 222.88.
  • Regular refers to a change in the function of human muscle cell enhanced binding factor 222.88, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological properties and functions of human muscle cell enhanced binding factor 222.88. Or changes in immune properties.
  • 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 muscle cell enhanced binding factor 222.88 using standard protein purification techniques.
  • the substantially pure human muscle cell enhanced binding factor 222. 88 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of human muscle cell enhanced binding factor 222. 88 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence C-T-G-A
  • complementary sequence G-A-C-T.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern imprinting or Nor thern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of 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., Madis on Wi s.). The MEGALIGN program can compare two or more sequences according to different methods, such as the Clus ter method (Hi gg ins, DG and PM Sharp (1988) Gene 73: 237-244). 0 The Cluster method checks all pairs The distances of each group are arranged into clusters. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences, such as sequence A and sequence B, is calculated by the following formula: The number of matching residues between the sequence and the sequence S
  • the percent identity between nucleic acid sequences can also be determined by the Cluster method or by methods known in the art such as Jotun He in (He in J., (1990) Me thods in enzymo l ogy 183: 625-645).
  • Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitution for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to HFP or a chemical modification of its nucleic acid. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and? 7. It can specifically bind to the epitope of human muscle cell enhanced binding factor 222.88.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not part of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances in the natural state .
  • isolated human muscle cell enhanced binding factor 222. 88 refers to human muscle cell enhanced binding factor 222. 88 and is substantially free of other proteins, lipids, carbohydrates, or other substances naturally associated with it.
  • Those skilled in the art can purify human muscle cell enhanced binding factor 222.88 using standard protein purification techniques.
  • Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel.
  • the purity of human muscle cell enhanced binding factor 222. 88 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a novel polypeptide-human muscle cell enhanced binding factor 222.88, which is basically composed of Consisting of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of human muscle cell enhanced binding factor 222.88.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human muscle cell enhanced binding factor 222.88 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 one or more amino acid residues are substituted with other groups to include a substituent; or (III) such A type in which a mature polypeptide is fused to another compound (such as a compound that extends the half-life of a polypeptide, such as polyethylene glycol); or (IV) a type of polypeptide sequence in which an additional amino acid sequence is fused into a 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 1367 bases in length and its open reading frame 425-1051 encodes 208 amino acids. According to the comparison of gene chip expression profiles, it was found that this peptide has a similar expression profile with human muscle cell enhanced binding factor 2, and it can be inferred that the human muscle cell enhanced binding factor 222. 88 has similar functions to human muscle cell enhanced binding factor 2.
  • the polynucleotide of the present invention may be in the DM form or the RM form.
  • DNA forms include cDNA, genomic DNA, or synthetic DM.
  • DNA can be single-stranded or double-stranded.
  • DM can be coded or non-coded.
  • 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 (and optional additional coding sequences) of the mature polypeptide and non-coding sequences.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising 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 variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity, between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • "strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.12SDS, 60 ° C; or (2) during hybridization Add denaturant, such as 50 ° /.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nuclei. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding human muscle cell enhanced binding factor 222.88.
  • 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 muscle cell enhanced binding factor 222.88 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DM is the least commonly used. Direct chemical synthesis of DM sequences is often the method of choice.
  • the more commonly used method is the isolation of cDNA sequences. Isolate cDNA of interest
  • the standard method is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • kits are also commercially available (Qi agene).
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manua 1, Cold 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.
  • 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-DNA or DM-RNA hybridization; (2) the presence or loss of marker gene function; (3) the level of human muscle cell enhanced binding factor 222.88 transcripts (4) Detecting protein products expressed by genes through immunological techniques or measuring biological activity. The above methods can be used alone or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably 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 herein is usually a DM sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of the human muscle cell enhanced binding factor 222.88 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • a method for amplifying DM / RNA by PCR is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be 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 cDM sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDM sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising a polynucleotide of the present invention, and a host cell genetically engineered using the vector of the present invention or directly using human muscle cell enhanced binding factor 222.88 coding sequence, and Method for producing a polypeptide of the present invention by recombinant technology.
  • a polynucleotide sequence encoding human muscle cell enhanced binding factor 222.88 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct an expression vector that combines the DNA sequence encoding human muscle cell enhanced binding factor 222.88 and appropriate transcriptional / translational 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 mMA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells.
  • Enhancers are cis-acting factors expressed by DM, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers and adenovirus enhancers on the late side of the origin of replication.
  • 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 muscle cell enhanced binding factor 222.88 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute the polynucleotide or the recombinant vector.
  • Genetically engineered host cells refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • 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 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 DM 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 recombinant human muscle cell enhanced binding factor 222. 88 (Scence, 1984; 224: 1431). Generally, the following steps are taken:
  • 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.
  • Muscle cell enhancement binding factor 2 is a protein and a variety of muscle-specific enhancers and promoters. Center of the important role of cooperation. Muscle-enhancing factors bind to the nucleus of muscle and brain cells in vivo to regulate the development and growth of relevant tissues in the body.
  • Myocyte-enhanced binding factor 2 constitutes an independent protein family in the body-the myocyte-enhanced binding factor 2 transcription factor protein family. Members of this protein family are highly expressed in skeletal muscle and brain. Stimulated expression.
  • MEF2C protein is a new member of the human muscle cell enhanced binding factor 2 protein family.
  • the protein is also stimulated in the late stages of muscle tissue differentiation and development, and it plays an important role in regulating muscle development and nerve development in organisms. Mutations or abnormal expression of the protein sequence fragments will directly lead to abnormal development of related muscle tissues and nervous systems, and then cause various related diseases.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of human hMEF2C protein, and both have similar biological functions.
  • the polypeptide of the present invention plays an important role in regulating muscle and nerve development in the body, and it can bind to specific binding factors in muscle cells or neurons to enhance biomolecular information feedback. Its abnormal expression is usually closely related to the occurrence of pathological processes such as muscular system development, abnormal development and differentiation of the nervous system, and tumors in related tissues, and cause related diseases.
  • the abnormal expression of the human muscle cell enhanced binding factor 222.88 of the present invention will produce various diseases, especially muscle diseases, neurological diseases, mental diseases, related tumors, and certain immune diseases. These diseases include but not limited to:
  • Muscle disorders muscular dysplasia, muscular dystrophy, myasthenia gravis, muscular pseudohypertrophy, Duchenne muscular dystrophy, tonic muscular dystrophy, myasthenia, bradykinesia, dystonia
  • Neurological diseases Degenerative diseases of the nervous system: Alzheimer's disease, Parkinson's disease, chorea, depression, amnesia, Huntington's disease, epilepsy, migraine, multiple sclerosis, neurocutaneous syndrome, peripheral nerves Disease, neural tube dysplasia, malformation of the brain, congenital hydrocephalus, congenital cerebral dysgenesis
  • Neuroblastoma Neuroblastoma, Astrocytoma, Ependymal Tumor, Glioblastoma, Neurofibromatosis, Muscle Tumor
  • Certain immune disorders systemic lupus erythematosus, rheumatoid arthritis, post-infection myocarditis, scleroderma, myasthenia gravis, Guillain-Barre syndrome, common variable immunodeficiency disease, primary B lymphocyte immunity Deficiency disease, acquired immunodeficiency syndrome
  • Abnormal expression of the human muscle cell-enhancing binding factor 222.88 of the present invention may also cause certain genetic diseases and the like.
  • polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases W
  • Therapy can treat various diseases, especially muscle diseases, neurological diseases, psychiatric diseases, related tumors, certain immune diseases, certain genetic diseases, etc.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human muscle cell enhanced binding factor 222.88.
  • Agonists enhance human muscle cell-enhancing binding factors 222. 88 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 muscle cell enhanced binding factor 222.88 can be cultured together with a labeled human muscle cell enhanced binding factor 222.88 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human muscle cell enhanced binding factor 222. 88 include antibodies, compounds, receptor deletions, and the like that have been screened.
  • the antagonist of human muscle cell enhanced binding factor 222.88 can bind to human muscle cell enhanced binding factor 222.88 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide to make the polypeptide Cannot perform biological functions.
  • human muscle cell enhanced binding factor 222.88 can be added to the bioanalytical assay by determining the effect of the compound on the interaction between human muscle cell enhanced binding factor 222.88 and its receptors. Determine if the compound is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same way as for screening compounds described above. Polypeptide molecules capable of binding to human muscle cell enhanced binding factor 222. 88 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, the human muscle cell enhanced binding factor 222. 88 molecule should generally be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against the human muscle cell enhanced binding factor 222.88 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 obtained by direct injection of human muscle cell enhanced binding factor 222.88 into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to 'S adjuvant and so on.
  • Techniques for preparing monoclonal antibodies to human muscle cell enhanced binding factor 222.88 include, but are not limited to, hybridoma technology (Kohler and Milste in. Nature, 1975, 256: 495-497), triple tumor technology, human B-cells 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 (US Pat No. 4946778) can also be used to produce single chain antibodies against human muscle cell enhanced binding factor.
  • Antibodies against human muscle cell enhanced binding factor 222. 88 can be used in immunohistochemical techniques to detect human muscle cell enhanced binding factor 222. 88 in biopsy specimens.
  • Monoclonal antibodies that bind to human muscle cell enhanced binding factor 222. 88 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.
  • High-affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP, and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human muscle cell enhanced binding factor 222. 88 Positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human muscle cell enhanced binding factor 222.88.
  • Administration of an appropriate dose of antibody can stimulate or block the production or activity of human muscle cell enhanced binding factor 222.88.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human muscle cell enhanced binding factor 222.88.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human muscle cell enhanced binding factor 222. 88 detected in the test can be used to explain the importance of human muscle cell enhanced binding factor 222. 88 in various diseases and to diagnose human muscle cell enhanced binding factor 222. 88. A working 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 human muscle cell enhanced binding factor 222.88 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 muscle cell enhanced binding factor 222.88.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutant human muscle cell enhanced binding factor 222.88 to inhibit endogenous human muscle cell enhanced binding factor 222.88 activity.
  • a variant human muscle cell enhanced binding factor 222.88 may be a shortened human muscle cell enhanced binding factor 222.88 that lacks a signaling functional domain. Although it can bind to downstream substrates, it lacks signal transduction. active.
  • the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human muscle cell enhanced binding factor 222.88.
  • 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 muscle cell enhanced binding factor 222.88 into a cell. Construction of a recombinant disease carrying a polynucleotide encoding human muscle cell enhanced binding factor 2 22. 88 The method of virulence vectors can be found in the existing literature (Sambrook, eta l.). In addition, a recombinant polynucleotide encoding human muscle cell enhanced binding factor 222.88 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 muscle cell enhanced binding factor 222. 88 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 MA for endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained using any existing RM 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 DM sequence encoding the MA. This DM sequence has been integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human muscle cell enhanced binding factor 222. 88 can be used for the diagnosis of diseases related to human muscle cell enhanced binding factor 222. 88.
  • Polynucleotide encoding human muscle cell enhanced binding factor 222. 88 can be used to detect the expression of human muscle cell enhanced binding factor 222. 88 or the abnormal expression of human muscle cell enhanced binding factor 222. 88 in a disease state.
  • the DNA sequence encoding human muscle cell enhanced binding factor 222. 88 can be used to hybridize biopsy specimens to determine the expression of human muscle cell enhanced binding factor 222. 88.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization.
  • polynucleotides of the present invention can be used as probes to be fixed on micro arrays or DNA chips (also known as "gene chips") for analyzing differential expression analysis of genes in tissues and genetic diagnosis.
  • 88 specific primers for RM-polymerase chain reaction (RT-PCR) amplification in vitro can also detect the transcription product of human muscle cell enhanced binding factor 222. 88.
  • Human muscle cell enhanced binding factor 222.88 gene can also be used to diagnose human muscle cell enhanced binding factor 222. 88-related diseases.
  • Human muscle cell enhanced binding factor 222.88 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild type human muscle cell enhanced binding factor 222.88 DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR, and in situ hybridization. In addition, mutations may affect protein expression. Therefore, Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • sequences of the invention are also valuable for chromosome identification. This sequence will be specific to someone The chromosome is in a specific location 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 labeling 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 D 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 difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the present invention also provides a kit or kit containing one or more containers, the containers containing one or more An ingredient of the pharmaceutical composition of the present invention.
  • the containers containing one or more An ingredient of the pharmaceutical composition of the present invention.
  • there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which reminders authorize them to be administered to humans by government agencies that manufacture, use, or sell them.
  • 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 muscle cell enhancing binding factor 222. 88 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dosage range of human muscle cell-enhancing binding factor 222.88 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 Isolat ion Kit (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA.
  • the Smart cDNA cloning kit purchased from Clontech was used to insert the 00 fragment into the multiple cloning site of pBSK (+) vector (Clontech) to transform DH5 ⁇ , and the bacteria formed a cDNA library.
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public DM sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0643F04 was new DNA.
  • a series of primers were synthesized to perform bidirectional determination of the inserted CDM fragments contained in this clone.
  • the total RM of fetal brain cells was used as a template, and oligo-dT was used as a primer for reverse transcription reaction to synthesize cDNA.
  • PCR amplification was performed with the following primers:
  • Primer 1 5
  • GGAGATTGCGGCGATGTCGCAGGG -3 5
  • SEQ ID NO: 3 5
  • Primer2 5,-AGATGAATAGGATTTATTAGTCAA -3, (SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp of the 5th end of SEQ ID NO: 1;
  • Primer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.
  • Amplification reaction conditions containing 5 0mmol / L KC1 in a reaction volume of 50 ⁇ 1, 10mmol / L Tris- HC1, pH8 5, 1. 5mmol / L MgCl 2, 200 ⁇ 1 / 1 dNTP, lOpmol primer, 1U of Taq DM. Polymerase (Clontech). The reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 55. C 30sec; 72 ° C 2min. During RT-PCR, set ⁇ -act in as a positive control and template blank as a negative control.
  • the amplified product was purified using a QIAGEN kit and ligated to a PCR vector using a TA cloning kit (Invitrogen).
  • the DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as l-1367bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of expression of human myocyte enhancer binding 222. 8 ⁇ factor gene Total RNA was extracted one step [Anal. Biochem 1987, 162, 156-159]. This method involves acid guanidinium thiocyanate phenol-chloroform extraction. I.e. with 4M guanidine isothiocyanate - 25 mM sodium citrate, 0. 23 ⁇ 41 sodium acetate (.
  • RNA pellet was washed with 70% ethanol, dried and dissolved in water. 2 ⁇ g RNA was performed on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (H7. 0) -5 mM sodium acetate-IraM EDTA-2. 2M formaldehyde electrophoresis then transferred to nitrocellulose by a -.
  • 32 P dATP Preparation 32 P- DNA probe labeled by the random primer SYSTEM DNA probes used for PCR amplification shown in FIG human myocyte enhancer. Binding factor 22 2. 88 coding region sequence (425bp to 1051bp). 32P-labeled probe (approximately 2 x 10 6 cpm / ml) was hybridized with RNA-transferred nitrocellulose membrane in a solution at 42 ° C. Overnight, the solution contained 50% formamide-25raM KH 2 P0 4 ( ⁇ 7.4) -5 ⁇ 3 (5 061111 ⁇ 21 (11;, 3 solution and 20 ( ⁇ 8/1111 salmon sperm 1 ⁇ human. After hybridization, The filter was washed in 1 x SSC-0.
  • Example 4 In vitro expression, isolation and purification of recombinant human muscle cell enhanced binding factor 222. 88 According to SEQ ID NO: 1 and the coding region sequence shown in FIG. 1, a pair of specific amplification primers was designed, and the sequences are as follows:
  • Pr imer3 5'-CATGCTAGCATGGATTTCTCGATTATTCAGTAT-3 '(Seq ID No: 5)
  • Primer4 5' -CCCAAGCTTTTAGGCCTTTTTCAATTTTTCTAT-3 '(Seq ID No: 6)
  • the 5 'ends of these two primers contain Nhel and Hindlll digestion sites, respectively, followed by the coding sequences of the 5' and 3 'ends of the target gene, respectively.
  • the Nhel and Hindl ll digestion sites correspond to the expression vector plasmid pET-28b. (+) Selective endonuclease site on (Product of Novagen, Cat. No. 69865. 3).
  • the PCR reaction was performed using the PBS-0643F04 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-0643F04 plasmid, primers Primer-3 and Primer-4 were lOpmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1, respectively. Cycle parameters: 94. C 20s, 60. C 30s, 68. C 2 min, a total of 25 cycles. Nhel and Hindlll were used to double-digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligation product was transformed into E. coli DH5c by the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 3 ( ⁇ g / ml)), the positive clones were screened by colony PCR method and sequenced. Positive sequence correct clone (pET-0643F04) was used to transform the recombinant plasmid into E. coli BL21 (DE3) plySs (product of Novagen) by calcium chloride method. Cultured in LB liquid containing kanamycin (final concentration 30 ⁇ ⁇ / ⁇ 1) In the medium, the host strain BL21 (pET-0643F04) was cultured at 37 ° C.
  • polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin For methods, see: Avrameas, et al. Immunochemis try, 1969; 6: 43.
  • 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 for use as hybridization probes should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • 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 For homology comparison of the regions, 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 generally;
  • 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 sample membrane was placed in a plastic bag, and 3-10 mg of prehybridization solution (10xDenhardt's; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DM)) was added. After sealing the bag, shake at 68 ° C for 2 hours.
  • prehybridization solution 10xDenhardt's; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DM)
  • Gene chip or gene microarray (DM Microarray) is currently in many national laboratories and large pharmaceutical The companies are starting to develop and develop new technology. It refers to the orderly and high-density arrangement of a large number of target gene fragments on sloped glass, silicon and other carriers, and then uses fluorescence detection and computer software to compare and analyze data. In order to achieve the purpose of fast, efficient and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases . The specific method steps have been reported in the literature.
  • a total of 4,000 polynucleotide sequences of various full-length cDMs are used as target DNA, including the polynucleotide of the present invention. They were respectively amplified by PCR. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 ⁇ . The spotted slides were hydrated, dried, and cross-linked in a UV cross-linking instrument. After elution, the DNA was fixed on the glass slide to prepare a chip. The specific method steps have been reported in the literature. The sample post-processing steps in this embodiment are:
  • Total raMA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and mRNA was purified using Oligotex mRNA Midi Kit (purchased from QiaGen).
  • Cy3dUTP (5-Araino-propargyl-2'-deoxyuridine 5 '-triphate coupled to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech) labeled mRM of human mixed tissue, using the fluorescent reagent Cy5dUTP (5-Amino-propargyl- 2' -deoxyuridine 5 ' ⁇ triphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech Company, labeled mRM of specific tissues (or stimulated cell lines) of the body, and the probes were prepared after purification.
  • Cy5dUTP (5-Amino-propargyl- 2' -deoxyuridine 5 ' ⁇ triphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia
  • 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. Scanning was then performed with a ScanArray 3000 scanner (purchased from General Scanning, USA). 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 bladder mucosa, PMA + Ecv304 cell line, LPS + Ecv304 cell line thymus, normal fibroblasts 1024NC, Fibroblas t, growth factor stimulation, 1024NT, and scar-like fc growth Factor stimulation, 1013HT, scar into fc without stimulation with growth factor, 1013HC, bladder cancer cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunum adenocarcinoma, cardia cancer . Based on these 17 Cy3 / Cy5 ratios, a histogram is drawn (Figure 1). It can be seen from the figure that the expression profiles of human muscle cell enhanced binding factor 222.88 and human muscle cell enhanced binding factor 2 according to the present invention are very similar.

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Abstract

L'invention concerne un nouveau polypeptide, un facteur humain 222.88 associé à la prolifération de myocytes, 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 dystrophies musculaires, de neuropathologies, de maladies psychologiques, des tumeurs apparentées, de certaines maladies immunitaires et de certaines maladies héréditaires. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant le facteur humain 222.88 associé à la prolifération de myocytes.
PCT/CN2001/000912 2000-06-07 2001-06-04 Nouveau polypeptide, facteur humain 222.88 associe a la proliferation de myocytes, et polynucleotide codant ce polypeptide WO2002026813A1 (fr)

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CN00116363.9 2000-06-07
CN 00116363 CN1326976A (zh) 2000-06-07 2000-06-07 一种新的多肽——人肌细胞增强结合因子222.88和编码这种多肽的多核苷酸

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998001553A2 (fr) * 1996-07-09 1998-01-15 Genetics Institute, Inc. Proteines secretees et polynucleotides codant lesdites proteines

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998001553A2 (fr) * 1996-07-09 1998-01-15 Genetics Institute, Inc. Proteines secretees et polynucleotides codant lesdites proteines

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] 4 February 2000 (2000-02-04), LE T. ET AL., retrieved from GI:2341016 accession no. NCBI Database accession no. (AC002485.1) *
DATABASE PROTEIN [online] 29 October 1999 (1999-10-29), CHOISNE N. ET AL., retrieved from GI:7487986 accession no. NCBI Database accession no. (T12975) *

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