[go: up one dir, main page]

WO2002026799A1 - Nouveau polypeptide, chaine intermediaire moyenne de proteine de motilite cytoplasmique humaine 9.35, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, chaine intermediaire moyenne de proteine de motilite cytoplasmique humaine 9.35, et polynucleotide codant ce polypeptide Download PDF

Info

Publication number
WO2002026799A1
WO2002026799A1 PCT/CN2001/000960 CN0100960W WO0226799A1 WO 2002026799 A1 WO2002026799 A1 WO 2002026799A1 CN 0100960 W CN0100960 W CN 0100960W WO 0226799 A1 WO0226799 A1 WO 0226799A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
polynucleotide
cytoplasmic dynein
intermediate chain
human cytoplasmic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2001/000960
Other languages
English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Biowindow Gene Development Inc
Original Assignee
Shanghai Biowindow Gene Development Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Biowindow Gene Development Inc filed Critical Shanghai Biowindow Gene Development Inc
Priority to AU2001289541A priority Critical patent/AU2001289541A1/en
Publication of WO2002026799A1 publication Critical patent/WO2002026799A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4716—Muscle proteins, e.g. myosin, actin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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, namely human cytoplasmic dynein intermediate chain (UC) 9.35, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique
  • Terminally oriented microtubule actuators (Paschal and Vallee, 1987), which are involved in retrograde axonal transport, afferent organelle movement, and some aspects of chromosome segregation. It is related to the components of various membrane organelles including endocytosis vesicles, lysosomes, and Golgi, and binds with microsomes in vivo, and also acts on negative terminal directed chromosomal movement. Antibodies to this complex can be used to label centromeres during mitosis (Pfarr et al., 1990; Steuer et al., 1990). During the early and middle stages of mitosis, cytokines are responsible for chromosome movement (Paschal. B.M., R.B. Vallee, (1987), Nature (Lond.), 330: 181-183).
  • Cytoplasmic dynein is a large multi-subdomain complex (Paschal et al., 1987), which is composed of two catalytic heavy chains (HCs), and its sub-subunits include several intermediate chains (ICs) and several Light intermediate chains (LICs), the middle chain of cytoplasmic dynein and the connection of dynein to the cell are related to centromere.
  • HCs catalytic heavy chains
  • LICs Light intermediate chains
  • IC-1 and IC-2 There are two known causes of the cytoplasmic dynein intermediate chain: IC-1 and IC-2, each encoding multiple isoforms due to multiple splicing mechanisms, at least two sites in IC-2 There is alternative splicing.
  • the second site is the same as the alternative splicing site in IC-1, and the selectable sequence at this site is closely related between IC-1 and IC-2.
  • the most consistent sequence between IC-1 and IC-2 is the half near the C-terminus. This half of the sequence has the function of binding to the catalytic heavy chain of dynein, and the N-terminal domain is rich in positively and negatively charged amino acids.
  • the coiled-coil domain, as well as a serine-rich cluster regulate phosphorylation of this region with microtubules.
  • Each IC isotype contains a series of "WD" repeats and templates for protein-to-protein signature sequences.
  • IC-1 is expressed only in brain tissue, while IC-2 is expressed in various human tissues (Kevin T. Vaughan, Richard B. Val lee, The Journal of Cell Biology, Vol. 131. No. 6, Part 1, (1995) 1507-1516).
  • bladder cancer construct cells EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell lines, placenta, spleen, prostate cancer, jejunum adenocarcinoma, and cardia cancer the expression profile of the polypeptide of the present invention is intermediate with human cytoplasmic dynein
  • the expression profiles of chains (IC) are very similar, so the functions of the two may be similar.
  • the present invention has been named human cytoplasmic dynein intermediate chain UC) 9. 35.
  • the human cytoplasmic dynein intermediate chain (IC) 9.35 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 identification in the art has been required More human cytoplasmic dynein intermediate chain (IC) 9.35 proteins involved in these processes, especially the amino acid sequence identification of this protein.
  • the separation of the new human cytoplasmic dynein intermediate chain (IC) 9.35 protein-coding gene also provides a 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 diseases, so it is important to isolate its coding DNA. Disclosure of invention
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding human cytoplasmic dynein intermediate chain (IC) 9.35.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human cytoplasmic dynein intermediate chain (IC) 9.35.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the polypeptide of the present invention-human cytoplasmic dynein intermediate chain (IC) 9.35.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with human cytoplasmic dynein intermediate chain (UC) 9.35 abnormalities.
  • the present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of: (a) a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID No. 2;
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 59-316 in SEQ ID NO: 1; and (b) a sequence having 1-577 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 present invention also relates to a screen for mimicking, activating, antagonizing or inhibiting human cytoplasmic dynein intermediate chains
  • IC A method of a 35-protein active compound comprising utilizing a polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the present invention also relates to a method for in vitro detection of a disease or disease susceptibility related to abnormal expression of human cytoplasmic dynein intermediate chain (IC) 9.35 protein, which comprises detecting the polypeptide or a polynucleotide sequence encoding the same in a biological sample. Mutations, or the amount or biological activity of a polypeptide of the invention in a biological sample.
  • IC cytoplasmic dynein intermediate chain
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the preparation of the polypeptide and / or polynucleotide of the present invention for the treatment of cancer, developmental disease or immune disease or other diseases caused by abnormal expression of human cytoplasmic dynein intermediate chain (IC) 9.35. Use of medicine.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a protein or polynucleotide “variant” refers to an amino acid sequence having one or more amino acids or nucleotide changes, or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, which The amino acid substituted in the amino acid has a structural or chemical property similar to that of the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response in appropriate animals or cells and to bind to specific antibodies.
  • An "agonist” refers to a molecule that, when combined with human cytoplasmic dynein intermediate chain UC) 9.35, can cause changes in the protein and thereby regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to the human cytoplasmic dynein intermediate chain (UC) 9.35.
  • Antagonist refers to a biological species that can block or regulate human cytoplasmic dynein intermediate chain (IC) 9.35 when combined with human cytosolic dynein intermediate chain UC) 9.35.
  • Active or immunologically active molecule can include proteins, nucleic acids, carbohydrates or any other molecule that can bind to human cytoplasmic dynein intermediate chain (IC) 9.35.
  • Regulation refers to a change in the function of human cytoplasmic dynein intermediate chain UC) 9.35, including an increase or decrease in protein activity, a change in binding characteristics, and any of the human cytoplasmic dynein intermediate chain UC) 9.35. Changes in other biological, functional or 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 the human cytoplasmic dynein intermediate chain (IC) using standard protein purification techniques.
  • a substantially pure human cytoplasmic dynein intermediate chain (IC) 9.35 produces a single main band on a non-reducing polyacrylamide gel.
  • Human cytoplasmic dynein intermediate chain (IC) 9. 35 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.
  • 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 achieved by hybridization under conditions of reduced stringency (Sou thern India Or Nor thern blot etc.) to detect.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other 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., Mad Son Wis.). 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 C lus ter method checks all The distances arrange the groups of sequences into clusters. The clusters are then assigned in pairs or groups.
  • the percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: The number of matching residues between sequence A and sequence X 100 The number of residues in sequence A-the number of spacer residues in sequence A The number of spacer residues in a sequence B can also be determined by the Cluster method or by a method known in the art such as Jotun He in (% in Hez J., (1990) Methods in emzurao logy 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.
  • Amino acids used for conservative substitutions may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and? ⁇ , which can specifically bind to the epitope of human cytoplasmic dynein intermediate chain UC) 9.35.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it occurs naturally).
  • a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not a component 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 cytoplasmic dynein intermediate chain UC) 9. 35 means human cytosolic dynein intermediate chain UC) 9. 35 is substantially free of other proteins, lipids, and sugars naturally associated with it. Class or other substances.
  • Those skilled in the art can purify the human cytoplasmic dynein intermediate chain (IC) 9.35 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human cytoplasmic dynein intermediate chain (IC) 9.35 peptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide-human cytoplasmic dynein intermediate chain UC) 9.35, which basically consists of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques.
  • polypeptide of the invention may be glycosylated, or it may be non-glycosylated.
  • the polypeptides of the invention may also include or exclude the initial methionine residue.
  • the invention also includes fragments, derivatives, and analogs of human cytoplasmic dynein intermediate chain (IC) 9.35.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human cytoplasmic dynein intermediate chain (IC) 9.35 of the present invention.
  • a fragment, derivative, or analog of the polypeptide of the present invention may be: (I) a type in which one or more amino acid residues are replaced 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 One, wherein the mature polypeptide is fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) such a polypeptide sequence in which an additional amino acid sequence is fused into the mature polypeptide ( Such as leader or secretory sequences or Sequences or protease sequences) As set forth herein, such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a CDM library of human fetal brain tissue. It contains a polynucleotide sequence that is 577 bases in length and its open reading frames 59-316 encode 85 amino acids.
  • this polypeptide has a similar expression profile with human cytoplasmic dynein intermediate chain UC), and it can be inferred that the human cytosolic dynein intermediate chain (IC) 9. 35 has a human cytosolic dynein intermediate Chain (IC) similar functions.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 in the present invention, but which differs from the coding region sequence shown in SEQ ID NO: 1.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the 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 invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% F i co ll, 42 ° C, etc .; or (3) only between the two sequences Crosses occur at least 95% or more, and more preferably 97% or more. and the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 cores. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding human cytoplasmic dynein intermediate chain (IC) 9.35.
  • 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 cytoplasmic dynein intermediate chain UC) 9.35 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) separating the double-stranded DNA sequence from the DM of the genome; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DM sequences is often the method of choice.
  • the more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • the construction of c; DNA 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.
  • the genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RM hybridization; (2) the presence or absence of marker gene functions; (3) determination of human cytoplasmic dynein intermediate chain (IC) 9:35 The level of transcripts; (4) Detecting protein products expressed by genes by immunological techniques or by measuring biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used herein is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention. The genes or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product for detecting human cytoplasmic dynein intermediate chain (IC) 9.35 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).
  • the RACE method RACE-cDM terminal rapid amplification method
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, the sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using a vector of the present invention or directly using human cytoplasmic dynein intermediate chain (UC) 9.35, and the production of the present invention by recombinant technology Said method of polypeptide.
  • a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using a vector of the present invention or directly using human cytoplasmic dynein intermediate chain (UC) 9.35, and the production of the present invention by recombinant technology Said method of polypeptide.
  • a polynucleotide sequence encoding human cytoplasmic dynein intermediate chain UC) 9.35 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.
  • IC cytoplasmic dynein intermediate chain
  • Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human cytoplasmic dynein intermediate chain (IC) 9.35 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989).
  • the DNA sequence can be effectively linked to an appropriate promoter in an expression vector, To guide raRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors 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 on the late side of the origin of replication, and adenovirus enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and Green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and Green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human cytoplasmic dynein intermediate chain UC) 9.35 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetic engineering containing the polynucleotide or the recombinant vector.
  • Host 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.
  • Escherichia coli, Streptomyces bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells insect cells
  • fly S2 or Sf9 animal cells
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of DNA uptake can be harvested after exponential growth phase, with (: Treatment 1 2, steps well known in the art used alternative is to use MgCl 2..
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposomes Packaging, etc.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human cytoplasmic dynein intermediate chain UC) 9.35 (Science, 1984; 224: 1431). Generally speaking, there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography
  • FIG. 1 is a comparison diagram of gene chip expression profiles of human cytoplasmic dynein intermediate chain (IC) 9.35 and human cytoplasmic dynein intermediate chain (IC) of the present invention.
  • the upper figure is a graph of the expression profile of human cytoplasmic dynein intermediate chain (IC) 9. 35, and the lower picture is the expression profile of the human cytoplasmic dynein intermediate chain (UC) 9.35.
  • 1-bladder mucosa 2- PMA + Ecv304 cell line, 3- LPS + Ecv304 cell line thymus, 4- normal fibroblast 1024NC; 5-Fibroblas t ;, growth factor stimulation, 1024NT, 6- scar into fc Growth factor stimulation, 101 3HT, 7- scar into fc without stimulation with growth factor, 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 is a polyacrylamide gel electrophoresis image of isolated human cytoplasmic dynein intermediate chain (IC) 9.35.
  • 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 cDM fragment into the multiple cloning site of pBSK (+) vector (Clontech) to transform DH5 ⁇ .
  • the bacteria formed a cDNA library.
  • Dye terminate cycle reaction ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with a public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0845B06 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • CDNA was synthesized using fetal brain total RNA as a template and ol igo-dT as a primer for reverse transcription reaction. After purification using Qiagene's kit, the following primers were used for PCR amplification:
  • Pr imerl 5'- GGGGACATCATTTTCACCCCGTTA -3 '(SEQ ID NO: 3)
  • Primer2 5'- GGCAAAAATACATTTTATTTATTA -3, (SEQ ID NO: 4)
  • Pr imerl is a forward sequence starting at the lbp at the 5 'end of SEQ ID NO: 1;
  • Pr imer2 is the 3'-end reverse sequence in SEQ ID NO: 1.
  • Amplification reaction conditions reaction volume containing 50 ⁇ 1 of the surface 50 ol / L KC1, 10mraol / L Tr i s- CI, (pH8 5.), 1. 5ramol / L MgCl 2, 200 ⁇ mol / L dNTP, l Opmol primer, 1U 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 2min.
  • 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 1-577bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human cytoplasmic dynein intermediate chain (IC) 9. 35 gene expression: Total RNA was extracted in one step [Anal. Biochem 1987, 162, 156-159]. The method includes acid sulfur Guanidinium cyanate phenol-chloroform extraction.
  • the tissue is homogenized with 4M guanidinium isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ), Mix and centrifuge. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • a 32P-labeled probe (approximately 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM H 2 P0 ( pH7.4)-5xSSC-5xDenhardt, s solution and 200 g / ml salmon sperm DNA. After hybridization, the filters were placed at 1 X SSC-0.1 ° /. Wash in SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 4 In vitro expression, isolation and purification of recombinant human cytoplasmic dynein intermediate chain (IC) 9.35
  • Primer3 5,-CCCCATATGATGAGCTTCTCTAGCAGAAGGCGC- 3, (Seq ID No: 5)
  • Primer 4 5,-CCCGAATTCTCACTGCTTCCCCTTTCTTTC- 3, (Seq ID No: 6)
  • the two ends of these two primers contain Ndel and EcoRI digestion sites, respectively.
  • the coding sequences of the 5 'and 3' ends of the gene of interest are followed, respectively.
  • the Ndel and EcoRI restriction sites correspond to the selectivity within the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Digestion site.
  • the pBS-0845B06 plasmid containing the full-length target gene was used as a template for the PCR reaction.
  • the PCR reaction conditions are as follows: a total volume of 50 ⁇ l contains 10 pg of pBS-0845B06 plasmid, primers Primer-3 and Primer-4, and j is lpmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1. Cycle parameters: 94. C 20s, 60 ° C 30s, 68. C 2 min, a total of 25 cycles. Ndel and EcoRI were used to double digest the amplified product and plasmid pET-28 (+), respectively, and the Tai fragments were recovered and ligated with T4 ligase. The ligation product was transformed into E. coli DH5a by the calcium chloride method.
  • Polypeptide synthesizer (product of PE company) was used to synthesize the following human cytoplasmic dynein intermediate chain (IC) 9. 35 specific peptides:
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin For the method, see: Avraraeas, et al. Immunocliemistry, 1969; 6: 43. Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once.
  • a titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum.
  • P harose total IgG isolated from the serum of rabbit antibodies with Protein A-Se.
  • the peptide was bound to a cyanogen bromide-activated Se P harose 4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography.
  • the immunoprecipitation method proved that the purified antibody could specifically bind to human cytoplasmic dynein intermediate chain (IC) 9.35.
  • Example 6 Application of the polynucleotide fragment of the present invention as a hybridization probe
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods. They all use the same steps of hybridization after fixing the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer, so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthetic polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps. This embodiment makes use of higher intensity membrane washing conditions (such as lower salt concentration and higher temperature) to enable hybridization
  • the 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
  • the GC content is 30% -70%, and the non-specific hybridization increases when it exceeds;
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutant sequence (41Nt) of the gene fragment of SEQ ID NO: 1 or its complementary fragment:
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • Two NC membranes are required for each probe, so that they can be used in the following experimental steps.
  • the film was washed with high-strength conditions and strength conditions, respectively.
  • the 32 P-Probe (the second peak is free ⁇ - 32 P-dATP) is prepared.
  • Gene microarray or DNA microarray is a new technology that many national laboratories and large pharmaceutical companies are working on. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass. , Silicon and other carriers, and then use fluorescence detection and computer software to compare and analyze the data, in order to achieve the purpose of rapid, efficient, high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen for tissue-specific new 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. , M., Cha i, A., Sha lom, 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. After the purified amplified product was purified, the concentration 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 the points is 280 ⁇ ⁇ . The spotted slides were hydrated, dried, and cross-linked in a purple diplomatic coupling instrument. After elution, the DNA was fixed on a glass slide to prepare a chip. The specific method steps have been reported in the literature in various ways. The post-spot processing steps of this embodiment are:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) by one-step method, and the mRNA was purified by Ol igotex mRNA Midi Kit (purchased from QiaGen), and separated by reverse transcription! Cytodextrin test Cy3dUTP (5-Amino-propargyl-2'-deoxyur idine 5--tr iphate coupled to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech) was used to label the mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP (5- Amino- propargyl- 2'- deoxyur idine 5'-tr iphate coupled to Cy5 f luorescent dye, purchased from Amersham Phamacia Biotech) to label mRNA of specific tissues (or stimulated cell lines) of the body, and prepare them after purification Probe.
  • Cy3dUTP 5-Amino-propargyl-2'-deoxyur idine
  • the probes from the above two tissues and the chip were respectively hybridized in a UniHyb TM Hybridinium Solution (purchased from TeleChera) hybridization solution for 16 hours, and the washing solution (1 SSC, 0.2% SDS) was used at room temperature. After washing, scanning was performed with a ScanArray 3000 scanner (purchased from Genera Scanning, USA), and the scanned images were analyzed by Imagene software (Biodi scovery, 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, scar-like fc growth factor Stimulation, 1013H 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. Draw a graph based on these Cy3 / Cy5 ratios. (figure 1 ) . It can be seen from the figure that the expression profile of human cytoplasmic dynein intermediate chain (UC) 9.35 and human cytosolic dynein intermediate chain (IC) according to the present invention are very similar. Industrial applicability
  • polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infections and immune diseases.
  • Cytoplasmic dynein is a negative terminal directional microtubule actuator that is involved in retrograde axonal transport, the movement into organelles, and some aspects of chromosome segregation. Cytoplasmic dynein is a large multi-subunit complex, and its subunits include several intermediate chains (ICs) and several light intermediate chains (LICs). To cells it is related to centromeres.
  • IC intermediate chain
  • LICs light intermediate chains
  • the expression profiles of (IC) proteins are consistent, and both have similar biological functions.
  • the abnormal expression of the polypeptide of the present invention in vivo can affect the physiological function of cytoplasmic dynein, and then lead to the occurrence of diseases of the nervous system (sympathetic nervous system and parasympathetic nervous system). These diseases include, but are not limited to:
  • arrhythmias such as early atrial, early ventricular, sinus tachycardia, supraventricular tachycardia, ventricular tachycardia, atrial flutter, atrial fibrillation, sinus bradycardia, sinus arrest,
  • Sinus syndrome indoor conduction block, etc .
  • CAD angina pectoris
  • myocardial infarction cardiovascular neurosis
  • acute heart failure chronic heart failure
  • HBP chronic heart failure
  • Pulmonary edema respiratory muscle paralysis, respiratory failure, bronchial asthma, etc .
  • Gastrointestinal neurosis Hydatid disease, psychogenic vomiting, neurogenic gas, anorexia nervosa, irritable bowel Provoke syndrome, etc .;
  • dysmenorrhea dysmenorrhea, glaucoma, visual impairment and multiple organ ischemic necrosis, such as renal necrosis (renal failure), liver necrosis, intestine
  • cytoplasmic dynein acts on chromosome movement. Therefore, the abnormal expression of human cytoplasmic dynein intermediate chain UC) protein in vivo can affect the formation of chromosomes in early embryonic development, and then lead to the development of various developmental disorders.
  • the polypeptide of the present invention and its antagonists, agonists and inhibitors can be directly used for the treatment of various diseases, especially diseases and the like.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of human cytoplasmic dynein intermediate chain (UC) protein, and the two have similar biological functions.
  • the abnormal expression of the polypeptide of the present invention in vivo can affect the physiological function of cytoplasmic dynein, and then lead to the development of various developmental disorders, including but not limited to:
  • These diseases include but are not limited to the following, such as: congenital abortion, cleft palate, facial cleft lip, cervical sac, cervical fistula, limb absentness, limb differentiation disorder, gastrointestinal atresia or stenosis, ileal diverticulum, umbilical fistula , Congenital umbilical hernia, congenital aganglion-free megacolon, laryngotracheal stenosis or atresia, tracheoesophageal fistula, hyaline membrane disease, congenital pulmonary cyst, atelectasis, polycystic kidney, ectopic kidney, horse telluride Double ureter, umbilical fistula, cryptorchidism, congenital inguinal hernia, double uterus, vaginal atresia, hypospadias, hermaphroditism, atrial septal defect, ventricular septal defect, abnormal arterial stem
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human cytoplasmic dynein intermediate chain (IC) 9.35.
  • Agonists enhance human cytoplasmic dynein intermediate chain (IC) 9.35 stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • the drug can be, or mammalian cells are human cytoplasmic dynein intermediate table chain (IC) 9.35 membrane preparation with labeled human cytoplasmic dynein intermediate chain (IC) 9. 35 -. From the culture. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human cytoplasmic dynein intermediate chain (UC) 9.35 include selected antibodies, compounds, receptor deletions, and the like. Antagonists of human cytoplasmic dynein intermediate chain (IC) 9.35 can bind to human cytoplasmic dynein intermediate chain (IC) 9.35 and eliminate its function, or inhibit the production of the polypeptide, or with the active site of the polypeptide Binding prevents the polypeptide from functioning biologically.
  • human cytoplasmic dynein intermediate chain UC) 9.35 can be added to the bioanalytical assay. Influence to determine if a compound is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to human cytoplasmic dynein intermediate chain (IC) 9.35 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. Screening, typically those responsible for cytoplasmic dynein intermediate chain (IC) 9. 35 labeled molecule.
  • 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 cytoplasmic dynein intermediate chain (UC) 9.35 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, and fragments produced by the Fab library.
  • Polyclonal antibodies can be produced using human cytoplasmic dynein intermediate chain UC) 9.35. It can be obtained by direct injection of immunized animals (such as rabbits, mice, rats, etc.). A variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant, etc. Techniques for preparing monoclonal antibodies to human cytoplasmic dynein intermediate chain (UC) 9.35 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human B-cell hybridoma Technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that combine human constant regions with non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851). 0 Existing techniques for producing single-chain antibodies (US Pat No. .4946778) can also be used to produce single chain antibodies against human cytoplasmic dynein intermediate chain UC) 9.35.
  • Antibodies against human cytoplasmic dynein intermediate chain (UC) 9.35 can be used in immunohistochemistry to detect human cytosolic dynein intermediate chain (IC) 9.35 in biopsy specimens.
  • Monoclonal antibodies that bind to human cytoplasmic dynein intermediate chain (IC) 9.35 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 human cytoplasmic dynein intermediate chain (IC) 9.3 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 the human cytoplasmic dynein intermediate chain (IC ) 9.35 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human cytoplasmic dynein intermediate chain (UC) 9.35.
  • the proper dose of antibody can stimulate or block the production or activity of human cytoplasmic dynein intermediate chain (IC) 9.35.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human cytoplasmic dynein intermediate chain (UC) 9.35 level.
  • These tests are well known in the art and include FISH assays and radioimmunoassays.
  • the human cytoplasmic dynein intermediate chain (UC) 9.35 level detected in the test can be used to explain human cytoplasmic motility.
  • 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 cytoplasmic dynein intermediate chain (UC) 9.35 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 cytoplasmic dynein intermediate chain (UC) 9.35.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human cytoplasmic dynein intermediate chain (UC) 9.35 to inhibit endogenous human cytosolic dynein intermediate chain (IC) 9.35 activity.
  • a variant human cytoplasmic dynein intermediate chain (IC) 9.35 may be a shortened human cytosolic dynein intermediate chain (IC) 9.35 that lacks a signaling domain, although it can bind to downstream substrates, However, it lacks signaling activity. Therefore, recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of human cytoplasmic dynein intermediate chain (UC) 9.35.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc.
  • a polynucleotide encoding human cytoplasmic dynein intermediate chain (IC) 9.35 can be used to transfer a polynucleotide encoding human cytoplasmic dynein intermediate chain (IC) 9.35 into cells.
  • Methods for constructing recombinant viral vectors carrying a polynucleotide encoding a human cytoplasmic dynein intermediate chain (IC) 9.35 can be found in existing literature (Sambrook, et al.).
  • recombinant polynucleotide encoding human cytoplasmic dynein intermediate chain (UC) 9.35 can be packaged into liposomes and transferred to cells Inside. ⁇
  • 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
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA and performs endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technology, such as solid-phase phosphate amide chemical synthesis to synthesize oligonucleotides.
  • 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 carrier's RNA polymerase promoter.
  • nucleic acid molecule 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 phosphorothioate or peptide bond instead of the phosphodiester bond is used for the ribonucleoside linkage.
  • the polynucleotide encoding human cytoplasmic dynein intermediate chain (UC) 9.35 can be used for the diagnosis of diseases related to human cytoplasmic dynein intermediate chain (IC) 9.35.
  • the polynucleotide encoding human cytoplasmic dynein intermediate chain (IC) 9.35 can be used to detect the expression of human cytoplasmic dynein intermediate chain (IC) 9.35 or the human cytoplasmic dynein intermediate in a disease state Strand UC) 9. 35 Aberrant expression.
  • the DNA sequence encoding human cytoplasmic kinetic protein intermediate chain (IC) 9.35 can be used to hybridize biopsy specimens to determine the expression of human cytoplasmic kinetic protein intermediate chain (IC) 9.35.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are all mature and open technologies, and related kits are commercially available.
  • a part or all of the polynucleotides of the present invention can be used as probes to be fixed on a micro array or a DNA chip (also called a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Human cytoplasmic dynein intermediate chain (UC) 9.35 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect the human cytosolic dynein intermediate chain (IC) 9.35 transcription product.
  • Human cytoplasmic dynein intermediate chain (IC) 9.35 mutations include point mutations, translocations, deletions, recombinations and any other mutations compared to normal wild-type human cytoplasmic dynein intermediate chain UC) 9.35 DNA sequences Exception, etc. Mutations can be detected using well-known techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect the expression of proteins. 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 be crossed with 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 DNA sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDM clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendelian Inherance in Man (available online with Johns Hopkins University Welch Medica l Library). Linkage analysis can then be used to determine the relationship between genes and diseases that are mapped to chromosomal regions.
  • the difference in cDM 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 by 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 cytoplasmic dynein intermediate chain UC) 9. 35 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dose range of human cytoplasmic dynein intermediate chain (IC) 9.35 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un nouveau polypeptide, une chaîne intermédiaire moyenne de protéine de motilité cytoplasmique humaine 9.35, 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 maladies affectant le système nerveux (du système nerveux sympathique et du système nerveux parasympathique) et des troubles du développement. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant la chaîne intermédiaire moyenne de protéine de motilité cytoplasmique humaine 9.35.
PCT/CN2001/000960 2000-06-14 2001-06-11 Nouveau polypeptide, chaine intermediaire moyenne de proteine de motilite cytoplasmique humaine 9.35, et polynucleotide codant ce polypeptide Ceased WO2002026799A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001289541A AU2001289541A1 (en) 2000-06-14 2001-06-11 A novel polypeptide -human cytoplasmic dynein intermediated chain (ic) 9.35 and the polynucleotide encoding said polypeptide

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN00116479A CN1328043A (zh) 2000-06-14 2000-06-14 一种新的多肽——人胞质动力蛋白中间链(ic)9.35和编码这种多肽的多核苷酸
CN00116479.1 2000-06-14

Publications (1)

Publication Number Publication Date
WO2002026799A1 true WO2002026799A1 (fr) 2002-04-04

Family

ID=4585883

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2001/000960 Ceased WO2002026799A1 (fr) 2000-06-14 2001-06-11 Nouveau polypeptide, chaine intermediaire moyenne de proteine de motilite cytoplasmique humaine 9.35, et polynucleotide codant ce polypeptide

Country Status (3)

Country Link
CN (1) CN1328043A (fr)
AU (1) AU2001289541A1 (fr)
WO (1) WO2002026799A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999014321A1 (fr) * 1997-09-17 1999-03-25 The Walter And Eliza Hall Institute Of Medical Research Nouvelles molecules therapeutiques
WO2000029846A2 (fr) * 1998-11-13 2000-05-25 Curagen Corporation COMPOSITIONS ET PROCEDES SE RAPPORTANT AUX MECANISMES D'ACTION DU RECEPTEUR-α ACTIVE PAR L'AGENT DE PROLIFERATION DU PEROXYSOME

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999014321A1 (fr) * 1997-09-17 1999-03-25 The Walter And Eliza Hall Institute Of Medical Research Nouvelles molecules therapeutiques
WO2000029846A2 (fr) * 1998-11-13 2000-05-25 Curagen Corporation COMPOSITIONS ET PROCEDES SE RAPPORTANT AUX MECANISMES D'ACTION DU RECEPTEUR-α ACTIVE PAR L'AGENT DE PROLIFERATION DU PEROXYSOME

Also Published As

Publication number Publication date
AU2001289541A1 (en) 2002-04-08
CN1328043A (zh) 2001-12-26

Similar Documents

Publication Publication Date Title
WO2002026799A1 (fr) Nouveau polypeptide, chaine intermediaire moyenne de proteine de motilite cytoplasmique humaine 9.35, et polynucleotide codant ce polypeptide
WO2002012320A1 (fr) Nouveau polypeptide, proteine humaine 12 liee a tnfr/ngfr contenant un domaine de cytochrome c, et polynucleotide codant ce polypeptide
WO2001090348A1 (fr) Nouveau polypeptide, chaine lourde de myoglobuline humaine 11, et polynucleotide codant ce polypeptide
WO2002012297A1 (fr) Nouveau polypeptide, proteine humaine 9 de liaison a la tropomoduline, et polynucleotide codant ce polypeptide
WO2001075048A2 (fr) Nouveau polypeptide, proteine ribosomale humaine s11 23, et polynucleotide codant pour ce polypeptide
WO2002012318A1 (fr) Nouveau polypeptide, glycosyl phosphatidylinositol polysaccharide f11.22, et polynucleotide codant ce polypeptide
WO2002002615A1 (fr) Nouveau polypeptide, cytochrome b5-9.57, et polynucleotide codant ce polypeptide
WO2001066584A1 (fr) Nouveau polypeptide, proteine humaine pax 9, et polynucleotide codant pour ce polypeptide
WO2002000823A2 (fr) Nouveau polypeptide, proteine d'activation enzymatique ras gtp 9.57, et polynucleotide codant ce polypeptide
WO2001081395A1 (fr) Nouveau polypeptide, adn topo-isomerase i-15, et polynucleotide codant pour ce polypeptide
WO2001090177A1 (fr) Nouveau polypeptide, activateur humain de la mort naturelle des cellules b13.64, et polynucleotide codant ce polypeptide
WO2002004633A1 (fr) Nouveau polypeptide, proteine humaine de fasciculation et d'extension 12.87, et polynucleotide codant ce polypeptide
WO2001083687A2 (fr) Nouveau polypeptide, proteine pax humaine 23, et polynucleotide codant pour ce polypeptide
WO2001068873A1 (fr) Nouveau polypeptide, molecule humaine d'adhesion intercellulaire 12, et polynucleotide codant pour ce polypeptide
WO2001092518A1 (fr) Nouveau polypeptide, proteine humaine 9.5 associee a la ccr4, et polynucleotide codant ce polypeptide
WO2002014365A1 (fr) Nouvelle proteine 12 activatrice du polypeptide-ras gtp et le polynucleotide codant pour ladite proteine
WO2002020595A1 (fr) Nouveau polypeptide, proteine a doigt de zinc humaine 18.04, et polynucleotide codant ce polypeptide
WO2002000831A2 (fr) Nouveau polypeptide, bromodomaine 12, et polynucleotide codant ce polypeptide
WO2002020580A1 (fr) Nouveau polypeptide, proteine humaine 15 tnfr/ngfr contenant un domaine de cytochrome c, et polynucleotide codant ce polypeptide
WO2001055419A1 (fr) Nouveau polypeptide, site de liaison 27 d'arn s1, et polynucleotide codant pour ce polypeptide
WO2001094532A2 (fr) Nouveau polypeptide, proteine ribosomale l39 9, et polynucleotide codant ce polypeptide
WO2002012322A1 (fr) Nouveau polypeptide, proteine humaine 12.32 de liaison de fk506, et polynucleotide codant ce polypeptide
WO2002006332A1 (fr) Nouveau polypeptide, proteine humaine de liaison de fk506, et polynucleotide codant ce polypeptide
WO2002020589A1 (fr) Nouveau polypeptide, proteine humaine associee aux tumeurs de la retine 19.91, et polynucleotide codant ce polypeptide
WO2001090172A1 (fr) Nouveau polypeptide, proteine ribosomale l39 13, et polynucleotide codant ce polypeptide

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CO CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP