WO2001075056A2 - Nouveau polypeptide, clathrine humaine a chaine legere 20, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, clathrine humaine a chaine legere 20, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001075056A2
WO2001075056A2 PCT/CN2001/000522 CN0100522W WO0175056A2 WO 2001075056 A2 WO2001075056 A2 WO 2001075056A2 CN 0100522 W CN0100522 W CN 0100522W WO 0175056 A2 WO0175056 A2 WO 0175056A2
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Prior art keywords
polypeptide
polynucleotide
light chain
clathrin light
sequence
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PCT/CN2001/000522
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English (en)
Chinese (zh)
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WO2001075056A3 (fr
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Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
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Priority to AU73778/01A priority Critical patent/AU7377801A/en
Publication of WO2001075056A2 publication Critical patent/WO2001075056A2/fr
Publication of WO2001075056A3 publication Critical patent/WO2001075056A3/fr

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • a new polypeptide one human clathrin light chain 20 and a polynucleotide encoding the polypeptide TECHNICAL FIELD
  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, a human clathrin light chain 20, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a method and application for preparing the polynucleotide and polypeptide. Background technique
  • phagocytosis ie, "cell swallowing"
  • puffing cell drinking
  • small Small particles small Small particles (diameter ⁇ 0.2 mu.m) were taken into the vesicles.
  • Puffing occurs most often at specific sites on the cell membrane called clathrin-coated four holes.
  • Clathrin is called a tripodin complex because of its three-legged appearance.
  • Clathrin-coated vesicles mediate selective transport of receptors from one cell membrane to another. They help receptor-mediated endocytosis and sorting proteins in the anti-Golgi network.
  • the clathrin tripod protein bodies self-assemble to form a closed polyhedral structure called a "cage."
  • clathrin's polyhedral assembly forms four holes that increase in the bend due to swallowing extracellular environment-derived substances.
  • Clathrin is a major membrane-forming protein that envelops vesicles that resemble envelope pockets and form cell surface fragments that participate in membrane traffic in integrated organisms.
  • the outer membrane of clathrin (known as the trigonelin complex) consists of three heavy chains (180Kd) and three light chains (from W to 27 Kd).
  • Clathrin's heavy chain provides the structural framework for the grid, forming a three-branch structure, while the light chain spans the internal fragments of each branch and can interact with other cytoplasmic proteins.
  • Formation of clathrin-coated vesicles requires the aggregation of tripodin complexes to a polyhedral network located on the cytoplasmic side of the cell membrane.
  • Clathrin assembly selectively traps receptors through its interaction with linker molecules, and eventually forms a spherical coating around the budding membrane vesicles. After re-emergence, clathrin is disassembled and vesicles are released to fuse with the target membrane. Multimolecular signals are required to adjust the time and location of clathrin on different cell inner membranes, to supplement clathrin subunits from the intracellular pool and to control clathrin disassembly.
  • Clathrin's light chain mediates these regulatory mechanisms, which may help to correctly orient and disassemble the clathrin outer membrane, non-covalently bind to the heavy chain, they also bind calcium and interact with the uncoated ATPase h S c70 interaction.
  • the two genes encode different but related light chains with 60% homology: LC (a) and LC (b). These two genes can produce two different forms through tissue-specific alternative splicing, and the two forms differ by inserting sequences of 30 or 18 residues, respectively.
  • Clathrin light chain linearly arranges 6 functional domains from N-terminus to C-terminus: phosphorylation targeting sequence; hs c70 junction Binding sequence; EF-hand-shaped calcium-binding sequence; 7-peptide repeat sequence with a coiled-coil ⁇ -helix; neuron-specific insertion sequence; C-terminal region with variable cysteine.
  • phosphorylation targeting sequence hs c70 junction Binding sequence
  • EF-hand-shaped calcium-binding sequence 7-peptide repeat sequence with a coiled-coil ⁇ -helix
  • neuron-specific insertion sequence C-terminal region with variable cysteine.
  • At the N-terminal portion of the clathrin light chain there is a region of 21 residues that is completely conserved in LC (a) and LC (b), which is acidic.
  • LC (b) LC
  • yeast there is a single light chain (gene CLC1) that is only approximately related to the light chain of higher organism
  • the first is a 7 amino acid peptide from the center of the conserved N-terminal region of the eukaryotic light chain.
  • the conserved sequence in the light chain of all higher eukaryotes FLAQ-Q-E-S; the second Derived from a positively charged region located at the C-terminus of all known clathrin light chains, conserved sequences in all organisms: [KR] -DXS- [KR]-[LIVM]-[KR] -x- [LIVM] (3) -xLK 0
  • Polypeptide containing the above-mentioned conservative peptide The antagonist of the polypeptide is an agonist inhibitor, which can diagnose and prevent and treat disorders related to abnormal vesicle transport.
  • human clathrin light chain 20 protein plays an important role in regulating important functions of the body, such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, so there has been a need to identify more involved in these The process of human clathrin light chain 20 protein, in particular, identifies the amino acid sequence of this protein. Isolation of the new clathrin light chain 20 protein encoding gene also provides a basis for the study 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 for DM. 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 an antibody directed against the polypeptide-to-human clathrin light chain 20 of the present invention.
  • Another object of the present invention is to provide analog compounds, antagonists, agonists, and inhibitors directed to the polypeptide-to-human clathrin light chain 20 of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities in human clathrin light chain 20.
  • 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 an amino acid sequence of SBQ 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) having SEQ ID NO: 1
  • the present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of human clathrin light chain 20 protein, which comprises utilizing the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for detecting a disease or disease susceptibility related to abnormal expression of human clathrin light chain 20 protein in vitro, which comprises detecting a mutation in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, or detecting The amount or biological activity of a polypeptide of the invention in a biological sample.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of human clathrin light chain 20.
  • Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein.
  • the following terms used in this specification and claims have the following meanings unless specifically stated: "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 DM 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 the 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 acid or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as the replacement of isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • an “insert” or “addition” 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.
  • Bioly active refers to a protein that has the structure, regulatory, 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 bound to human clathrin light chain 20, causes a change in the protein to regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that binds human clathrin light chain 20.
  • Antagonist refers to a molecule that can block or regulate the biological or immunological activity of human clathrin light chain 20 when bound to human clathrin light chain 20.
  • Antagonists and inhibitors can include proteins, nucleic acids, carbohydrates, or any other molecule that can bind human clathrin light chain 20.
  • Regulation refers to a change in the function of human clathrin light chain 20, including an increase or decrease in protein activity, a change in binding properties, and any other biological, functional, or immune properties of human clathrin light chain 20. change.
  • Substantially pure ' 1 means essentially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify human clathrin light chains using standard protein purification techniques.
  • the substantially pure human clathrin light chain 20 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of the human clathrin light chain 20 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to a polynucleotide that naturally binds by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence "C-T-G-A” can be combined with the complementary sequence "G-A-C-T”.
  • the complementarity between two single-stranded molecules can be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern or Northern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of a completely homologous sequence to a target sequence under conditions of reduced stringency. This does not mean that conditions with reduced stringency allow non-specific binding, because conditions with reduced stringency require that the two sequences interact with each other specifically or selectively.
  • 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 through the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Clus ter method (Higg ins, D. G. and P. M. Sharp (1988) Gene 73: 237-244). The Clus ter method arranges groups of groups into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: The number of matching residues between sequence A and sequence B
  • the number of residues in sequence A-the number of spacer residues in sequence A-the number of spacer residues in sequence B can also be determined by the Clus ter method or by methods known in the art such as Jotun Hein (He in J., (1990) Methods in erazumo 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; have uncharged heads
  • Amino acids with similar hydrophilic groups can 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 DM or RM sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to the “sense strand”.
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical 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 characteristics of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa, F (ab ') 2 and Fv, which can specifically bind to the epitope of human clathrin light chain 20.
  • 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 part of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state. .
  • isolated human clathrin light chain 20 means that human clathrin light chain 20 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify human clathrin light chain 20 using standard protein purification techniques. Substantially pure polypeptides produce a single main band on a non-reducing polyacrylamide gel. The purity of the human clathrin light chain 20 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human clathrin light chain 20, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be 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 It is glycosylated or 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 clathrin light chain 20.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the human clathrin light chain 20 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 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 the genetic code; or (II) such a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or (in) 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 the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protease sequence) As explained 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 a 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 1652 bases in length and its open reading frame of 1048-1593 encodes 181 amino acids.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cMA, genomic MA, or synthetic MA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a “degenerate variant” refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 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 that includes the polypeptide and a polynucleotide that includes additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be naturally occurring Allelic or non-naturally occurring variants.
  • These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (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 0.2 (SSC, 0.1 ° /. SDS, 60 ports; or (2) Add a denaturant during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.
  • the identity is at least 95%, and more preferably 97 ° /.
  • the hybridization occurs only when the above.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function as the mature polypeptide shown by SBQ ID NO: 2 And active.
  • 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 above. Nucleic acid fragments can also be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding human clathrin light chain 20.
  • 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 clathrin light chain 20 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 a double-stranded DM sequence from genomic DNA; 2) chemically synthesizing a MA sequence to obtain a double-stranded MA of the polypeptide.
  • genomic MA is the least commonly used. Direct chemical synthesis of DM sequences is the method of choice. The more commonly used method is the separation of cDM 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 cMA library. There are many mature techniques for mRNA extraction. Kits are also commercially available (Qiagene).
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecluar Cloning, A Laboratory Manual, Colling Harbor Laboratory. New York, 1989).
  • Commercially available cMA libraries are also available, such as different cMA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes can be screened from these cDNA libraries by conventional methods. These methods include (but not (Limited to): (l) DNA-DNA or DM-MA hybridization; (2) appearance or loss of marker gene function; (3) determination of transcript levels of human clathrin light chain 20; (4) through immunological techniques Or measure biological activity to detect protein products expressed by genes. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is usually 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.
  • MA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELI SA) can be used to detect the protein products expressed by the human clathrin light chain 20 gene expression.
  • ELI SA enzyme-linked immunosorbent assay
  • 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 cDNA 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 cMA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell that is genetically engineered using the vector of the present invention or directly using the human clathrin light chain 20 coding sequence, and a recombinant technology for producing the polypeptide of the present invention. method.
  • a polynucleotide sequence encoding the human clathrin light chain 20 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 expression vector It is characterized by containing replication origins, promoters, marker genes and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human clathrin light chain 20 and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DM technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manua 1, cold Spring Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or tr P promoter of E.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenoviral enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human clathrin light chain 20 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as insect cells such as Fly S2 or Sf9
  • 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 MA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human clathrin light chain 20 (Sc ience, 1984; 224: 1431). Generally there are the following steps: (1). Use the polynucleotide (or variant) encoding the human human clathrin light chain 20 of the present invention, or use a recombinant expression vector containing the polynucleotide to transform or transduce it appropriately Host cell
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography
  • FIG. 1 is a comparison diagram of gene chip expression profiles of human clathrin light chain 20 and human clathrin light chain 12 of the present invention.
  • the upper graph is a graph of the expression profile of human clathrin light chain 20, and the lower graph is the graph of the expression profile of human clathrin light chain 12.
  • 1 indicates fetal kidney
  • 2 indicates fetal large intestine
  • 3 indicates fetal small intestine
  • 4 indicates fetal muscle
  • 5 indicates fetal brain
  • 6 indicates fetal bladder
  • 7 indicates unstarved L02
  • 8 indicates L02 +, lhr, As 3+
  • 9 indicates ECV304 PMA-
  • 10 means ECV304 PMA +
  • 11 means fetal liver
  • 12 means normal liver
  • 13 means thyroid
  • 14 means skin
  • 15 means fetal lung
  • 16 means lung
  • 17 means lung cancer
  • 18 means fetal spleen
  • 19 means spleen
  • 20 Indicates prostate
  • 21 indicates fetal heart
  • 22 indicates heart
  • 23 indicates muscle
  • 24 indicates testis
  • 25 indicates fetal thymus
  • 26 indicates thymus.
  • FIG. 2 is a polyacrylamide gel electrophoresis image (SDS-PAGE) of an isolated human clathrin light chain 20.
  • FIG. 20kDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Human fetal brain total MA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using Quik mRNA Isolat ion Kit (product of Qiegene). 2ug poly (A) mRNA is cMA by reverse transcription. Using a Smart cDM cloning kit (purchased from Clontech), a 00 human fragment was inserted into a multicloning site of a pBSK (+) vector (Clontech) to transform DH5 ⁇ to form a cMA 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 MA sequence database (Genebank), and it was found that the CDM sequence of one of the clones 0162el0 was a new DNA.
  • a series of primers were synthesized to perform bidirectional determination of the inserted CDM fragments contained in this clone.
  • the 0162el0 clone contains a full-length cDNA of 1652bp (as shown by Seq ID N0: l), and has a 545bp open reading frame (0RF) from 1048bp to 1593bp, encoding a new protein (such as Seq ID NO : Shown in 2).
  • This cloned P BS- 0162el0 encoding a protein named human clathrin light chain 20.
  • Example 2 Cloning of a gene encoding human clathrin light chain 20 by RT-PCR
  • CDNA was synthesized using fetal brain cell total MA as a template and ol igo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, the following primers were used for PCR amplification:
  • Pr imer 1 5'- GGGGGTGGCGGTCTTTGGATTGGA-3 '(SEQ ID NO: 3)
  • Pr imer2 5'- CTTGCACAAAACAAAAATTTATTG-3 '(SEQ ID NO: 4)
  • Pr imerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Pr imer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.
  • Amplification reaction conditions 50 leg ol / L KC1, 10 mmol / L Tr is- CI, (pH 8.5.5), 1.5 mmol / L MgCl 2 , 200 ⁇ mol / L dNTP in a reaction volume of 50 ⁇ 1 , l Opmol primer, 1U Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DM thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min. in-? When 0! Was set, (3 0 ⁇ 11 was the positive control and the template blank was the negative control.
  • RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] 0
  • This method involves acid guanidinium thiocyanate-chloroform extraction. That is, the tissue was homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1), centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The obtained MA precipitate was washed with 70% ethanol, dried and dissolved in water.
  • the 32P- labeled probe (approximately 2 ⁇ 10 6 cpm / ml) and transferred to a nitrocellulose membrane RM 42 D C hybridized overnight in a solution, the solution comprising 50% formamide - 25mM KH 2 P0 4 (pH7. 4)-5 x SSC- 5 x Denhardt's solution and 200 ⁇ ⁇ / ⁇ 1 salmon essence DM. After hybridization, the filter was washed in 1 x SSC-0.1% SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 4 In vitro expression, isolation and purification of recombinant human clathrin light chain 20
  • Pr imer 3 5'- CCCCATATGATGAGTTTGTCCTTTCAGCCTAAG-3 '(Seq ID No: 5)
  • Primer 4 5 a CATGGATCCTCAGGAATCCTCAATTTTAGCTCT-3 '(Seq ID No: 6)
  • the 5 and 5 ends of these two primers contain Mel and BamHI digestion sites, respectively, followed by the coding sequences of the 5' and 3 'ends of the target gene, respectively.
  • the Ndel and BamHI restriction sites correspond to the selective endonuclease sites on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865. 3).
  • PCR was performed using the pBS-0162el0 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- 0162el 0 plasmid, primers Primer-3 and Primer- 4 points, and l) 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 BamHI were used to double-digest the amplified product and plasmid P ET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into the colibacillus DH5 0C by the calcium chloride method.
  • the titer of antibodies in rabbit serum was determined by BLISA using a 15 g / ml bovine serum albumin peptide complex-coated titer plate. Protein A-Sepharose was used to isolate total IgG from antibody-positive rabbit serum. The peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography. The immunoprecipitation method demonstrated that the purified antibody specifically binds to human clathrin light chain 20. '' 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 loaded And synthetic polymers.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SBQ ID 1 of the present invention; the second type of probes are partially the same as the multi-core of the present invention. Oligonucleotide SEQ ID NO: 1 Identical 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 for use as hybridization probes from the polynucleotide SEQ ID NO: 1 of the present invention 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 requirements can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 which belongs to the second type of probe, is equivalent to the replacement mutant sequence of the gene fragment or its complementary fragment (41Nt) of SEQ ID NO: 1:
  • 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) was added.
  • CT DM calf thymus DNA
  • Gene microarrays or DNA microarrays are new technologies currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon, and then fluorescence detection and computer software are used to achieve 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. For example, see DeRis i, JL, Lyer, V. & Brown, P. 0. (1997) Science 278, 680-686. And Hel le, RA, Schema, M., Chai, A., Shalom, D., (1997) PNAS 94: 2150-2155.
  • a total of 4,000 polynucleotide sequences of various full-length cDM are used as target DNA, including the polynucleotides 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 using a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 ⁇ m. The spotted slides are hydrated, dried, and placed in purple Cross-link in Diplomatic Instrument. After elution, the DNA is fixed on a glass slide to prepare a chip. The specific method steps are widely reported in the literature. The post-spot processing steps of this embodiment are:
  • 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), and the scanned images were analyzed by Iraagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, L02 cell line stimulated by arsenic for 1 hour, L02 cell line stimulated by arsenic for 6 hours prostate, heart, lung cancer, fetal bladder, fetal small intestine, fetal large intestine, fetal thymus, fetal muscle, fetal liver, fetal kidney, fetal spleen, fetal brain, Fetal lung And the fetal heart.
  • 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.
  • phagocytosis There are two main internal mechanisms of cellular uptake: phagocytosis and puffing. Puffing occurs most often at specific sites in the cell membrane called clathrin-coated cavities. Clathrin-coated vesicles mediate selective transport of receptors from one cell membrane to another. They contribute to receptor-mediated endocytosis and sorting proteins in the anti-Golgi network.
  • the outer membrane of clathrin is composed of three heavy chains and three light chains. Clathrin's heavy chain provides the structural framework for the grid, while the light chain spans the internal fragments of each branch and is able to interact with other cytoplasmic proteins.
  • Clathrin assembly selectively traps receptors through its interaction with linker molecules, and eventually forms a spherical coating around the budding membrane vesicles. After re-emergence, clathrin is disassembled and vesicles are released to fuse with the target membrane. Multimolecular signals are required to adjust the time and location of clathrin on different cell inner membranes, to supplement clathrin subunits from the intracellular pool, and to control clathrin disassembly. Clathrin's light chain mediates these regulatory mechanisms. It may help to correctly orient and disassemble the clathrin outer membrane. It is non-covalently bound to the heavy chain. They also bind calcium and interact with the uncoated ATPase hsc70. effect. Clathrin light chain-specific conserved sequences are required to form its active mot if.
  • the abnormal expression of the specific clathrin light chain mot if will cause the function of the polypeptide containing the mot if of the present invention to be abnormal, thereby causing abnormalities in vesicle-mediated selective receptor transport and further affecting the cells.
  • the transfer of information is related to the metabolism of the substance, causing diseases related to the degeneration of the nervous system, tumors, growth and development, and inflammation.
  • the abnormal expression of the human clathrin light chain 20 of the present invention will produce various diseases, especially degenerative changes of the nervous system, tumors, disorders of growth and development, and inflammation. These diseases include, but are not limited to:
  • Nervous system degenerative diseases Alzheimer's disease, Parkinson's disease, chorea, depression, amnesia, Huntington's disease, epilepsy, migraine, dementia, multiple sclerosis
  • tumors gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioblastoma, colon cancer , Melanoma, adrenal cancer, bladder cancer, bone cancer, osteosarcoma, myeloma, bone marrow cancer, brain cancer, uterine cancer, endometrial cancer, cholangiocarcinoma, colon cancer, thymus tumor, nasal cavity and sinus cancer, nasopharyngeal cancer
  • Inflammation allergic reaction, bronchial asthma, allergic pneumonia, adult respiratory distress syndrome, sarcoidosis, rheumatoid arthritis, rheumatoid arthritis, osteoarthritis, cholecystitis, glomerulonephritis, immune complex Types of glomerulonephritis, acute anterior uveitis, dermatomyositis, urticaria, atopic dermatitis, hemochromatosis, polymyositis, Addison's disease, chronic active hepatitis, emergency bowel syndrome, atrophy Gastritis, systemic lupus erythematosus, myasthenia gravis, cerebrospinal spinal multiple sclerosis, Guillain-Barre syndrome, intracranial granuloma, pancreatitis, myocarditis, and inflammation caused by infections and trauma
  • the abnormal expression of the human clathrin light chain 20 of the present invention will also produce certain hereditary, hematological and immune system diseases.
  • the polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially the degenerative changes of the nervous system, tumors, growth disorders, inflammation, and some Hereditary, hematological and immune system diseases.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human reticulin light chain 20.
  • Agonists enhance human clathrin light chain 20 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing human clathrin light chain 20 can be cultured together with labeled human clathrin light chain 20 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human clathrin light chain 20 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonist of human clathrin light chain 20 can bind to human clathrin light chain 20 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot function biologically. Learn function.
  • human clathrin light chain 20 can be added to a bioanalytical assay to determine whether a compound is a compound by measuring the effect of the compound on the interaction between human clathrin light chain 20 and its receptor.
  • 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 clathrin light chain 20 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. In screening, 20 molecules of human clathrin light chain should generally be marked.
  • the present invention provides polypeptides, and fragments, derivatives, analogs or cells thereof as antigens To produce antibodies.
  • antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against human clathrin light chain 20 epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human clathrin light chain 20 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's Agent.
  • Techniques for preparing monoclonal antibodies to human clathrin light chain 20 include, but are not limited to, hybridoma technology (Ohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma Technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions to 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 can also be used to produce single chain antibodies against human clathrin light chain 20.
  • Antibodies against human clathrin light chain 20 can be used in immunohistochemical techniques to detect human clathrin light chain 20 in biopsy specimens.
  • Monoclonal antibodies that bind to human clathrin light chain 20 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis. ⁇
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • human clathrin light chain 20 high affinity monoclonal antibodies can covalently bind to bacterial or phytotoxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a 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 clathrin light chain 20 positive cell.
  • the antibodies of the present invention can be used to treat or prevent diseases associated with human clathrin light chain 20.
  • Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human clathrin light chain 20.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human clathrin light chain 20 levels.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human clathrin light chain 20 detected in the test can be used to explain the importance of human clathrin light chain 20 in various diseases and to diagnose diseases in which human clathrin light chain 20 plays a role. .
  • 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 clathrin light chain 20 can also be used for a variety of therapeutic purposes. Gene therapy technology It can be used to treat abnormal cell proliferation, development or metabolism caused by the non-expression or abnormal / inactive expression of human clathrin light chain 20.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human clathrin light chain 20 to inhibit endogenous human clathrin light chain 20 activity.
  • a mutated human clathrin light chain 20 can be a shortened human clathrin light chain 20 lacking a signaling domain, and although it can bind to a downstream substrate, it lacks signaling activity.
  • the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of human clathrin light chain 20.
  • 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 clathrin light chain 20 into a cell.
  • Methods for constructing recombinant viral vectors carrying a polynucleotide encoding human clathrin light chain 20 can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human clathrin light chain 20 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: injecting the polynucleotide directly into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RM and DNA
  • ribozymes that inhibit human clathrin light chain 20 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RM molecule that can specifically decompose a specific MA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target MA and performs endonucleation.
  • Antisense RM, DM, and ribozymes can be obtained by any existing RM or DNA synthesis technology, such as solid-phase phosphoramidite synthesis method for oligonucleotide synthesis. Widely used.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RM. This DNA sequence is integrated downstream of the vector's RNA polymerase promoter. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphorothioate or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human clathrin light chain 20 can be used for the diagnosis of diseases related to human clathrin light chain 20.
  • the polynucleotide encoding human clathrin light chain 20 can be used to detect the expression of human clathrin light chain 20 or the abnormal expression of human clathrin light chain 20 in a disease state.
  • the DNA sequence encoding human clathrin light chain 20 can be used to hybridize biopsy specimens to determine the expression status of human clathrin light chain 20.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • 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 known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • RNA-polymerase chain reaction RT-PCR
  • RT-PCR RNA-polymerase chain reaction
  • Transcription products of human clathrin light chain 20 can be detected.
  • Detection of mutations in the human clathrin light chain 20 gene can also be used to diagnose human clathrin light chain 20-related diseases.
  • the forms of human clathrin light chain 20 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human clathrin light chain 20 DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for target chromosome locations.
  • an important first step is to locate these DM sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared based on cMA, 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 cMA libraries. '
  • CDM cloning with metaphase chromosomes by in situ hybridization allows precise chromosomal localization in one step.
  • FISH in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendel ian Inheritance in Man (available online with Johns Hopk ins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that are mapped to chromosomal regions.
  • the CDM or genomic sequence differences between the affected and the affected individuals need to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing diseased and unaffected individuals usually involves first looking for structural changes in the chromosome, such as defects visible at the chromosomal level or detectable with cDM sequence-based PCR Missing or transposing. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human clathrin light chain 20 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dosage range of human clathrin light chain 20 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

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Abstract

L'invention concerne un nouveau polypeptide, une clathrine humaine à chaîne légère 20, et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la clathrine humaine à chaîne légère 20.
PCT/CN2001/000522 2000-03-29 2001-03-26 Nouveau polypeptide, clathrine humaine a chaine legere 20, et polynucleotide codant pour ce polypeptide WO2001075056A2 (fr)

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CN 00115298 CN1315457A (zh) 2000-03-29 2000-03-29 一种新的多肽——人网格蛋白轻链20和编码这种多肽的多核苷酸
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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [Online] 06 November 1998 BIRREN B. ET AL. Database accession no. (AC005837) *
DATABASE GENBANK [Online] 21 October 1994 LONGSHORE J.W. Database accession no. (U15426) *
GENOME RES. vol. 8, no. 11, November 1998, pages 1097 - 1108 *
STAMM S. ET AL. NUCLEIC ACIDS RES. vol. 20, no. 19, 11 October 1992, pages 5097 - 5103 *

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