WO2002000829A2 - Nouveau polypeptide, proteine humaine 16.83 ftsh, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, proteine humaine 16.83 ftsh, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002000829A2
WO2002000829A2 PCT/CN2001/000946 CN0100946W WO0200829A2 WO 2002000829 A2 WO2002000829 A2 WO 2002000829A2 CN 0100946 W CN0100946 W CN 0100946W WO 0200829 A2 WO0200829 A2 WO 0200829A2
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polypeptide
polynucleotide
protein
human
sequence
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PCT/CN2001/000946
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Chinese (zh)
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WO2002000829A3 (fr
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Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
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Priority to AU89534/01A priority Critical patent/AU8953401A/en
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Publication of WO2002000829A3 publication Critical patent/WO2002000829A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide—a human ftsH protein 16.83 ′, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a method and application for preparing the polynucleotide and polypeptide. Background technique
  • ftsH protein is one of them. The study found that the distribution of ftsH protein is very extensive. People have found various members of the ftsH protein family in prokaryotes and some eukaryotes. The members of this protein family are highly conservative in evolution. They are all involved in regulating the division and growth of various tissues in the organism. Mutations or abnormal expressions will cause abnormal development and growth of related tissues in the body, and then trigger various related metabolism and development. Disorders and related tissue malignancies and cancers.
  • ftsH protein family Many members of the ftsH protein family have been cloned from mitochondria and chloroplasts of algae and some lower organisms.
  • Ryuuichi et al. Cloned a new member of the ftsH protein family, a tsH2 protein, from red algae.
  • the protein has similar structural and functional characteristics to known members of the ftsH protein family.
  • the protein sequence contains multiple conserved transmembrane structural regions and two transmembrane domains.
  • the C-terminus of the amino acid sequence of the protein is also It contains a highly conserved ATP-binding crust domain A and ATP-binding domain B, of which Haihan has an AAA domain.
  • ATP-binding crust domain may be an important active center for proteins to play ATPase.
  • protein sequence of the protein also contains a zinc finger binding site, a HEXXH scab domain, which may be an important active center of the protein for metalloproteinase activity.
  • the present invention has been named human ftsH protein 1 6.83.
  • the human ftsH protein 16.83 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 in the art to identify more people involved in these processes ftsH protein 16.83 protein, especially the amino acid sequence of this protein. Isolation of the newcomer ftsH protein 16.83 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 agents for disease 1 and it is therefore 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 a human nsH protein 16.83.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human ftsH protein 16.83.
  • Another object of the present invention is to provide a method for producing human ftsH protein 16.83.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention-human ftsH protein 16.83.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention-human ftsH protein 16.83.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities of human ftsH protein 16.83.
  • the present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 521-2982 in SEQ ID NO: 1; and (b) a sequence having 1-2249 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 method for screening compounds that mimic, activate, antagonize or inhibit the activity of human ft s H protein 16.83 protein, which comprises using the polypeptide of the present invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for in vitro detection of a disease or susceptibility to disease associated with abnormal expression of human ftsH protein 16.83 protein, 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 f t sH protein 16.83.
  • Nucleic acid sequence refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and may also refer to the genome or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof Minute.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a “variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion 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 and to bind to specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that, when combined with human f t s H protein 16.83, causes the protein to change, thereby regulating the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to human f t sH protein 16.83.
  • Antagonist refers to a molecule that can block or regulate the biological or immunological activity of human f t sH protein 16. 83 when combined with human f t sH protein 16. S 3. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind human f t sH protein 16.83.
  • Regular refers to a change in the function of human ft sH protein 16.83, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological, functional, or immune properties of human ft sH protein 16.83. change.
  • “Substantially pure '” 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 ft sH protein 16.83 using standard protein purification techniques. Basic The pure human f tsH protein 16.83 can generate a single main band on a non-reducing polyacrylamide gel. The purity of human fts H protein 16.83 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-GA
  • G-ACT complementary sequence
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands The efficiency and strength of hybridization between nucleic acid strands has a significant effect.
  • “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 blotting or Nor thern 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 the same or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Clus ter method (Hi gg 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.
  • sequence A and sequence B The percent identity between two amino acid sequences, such as sequence A and sequence B, is calculated by the following formula: Number of residues matching between sequence A and sequence X 100 Number of residues in sequence A-number of interval residues in sequence A
  • the number of spacer residues in a sequence B can also be determined by Cluster's method or using methods known in the art such as Jo tun He in (%). He He J. (1990) Methods in erazumo l ogy 183: 625-645).
  • Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitutions for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to 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 properties 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 ftsH protein 16.83.
  • 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 matter from its original environment (for example, its natural environment if it is naturally occurring).
  • a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a vector, or such a polynucleotide or polypeptide may be part of a 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 existing in the natural state. .
  • isolated human ftsH protein 16.83 means that human ftsH protein 16.83 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 ftsH protein 16.83 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the human ftsH protein 16.83 peptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human ftsH protein 16.83, 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 may be naturally purified products or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of human ftsH protein 16.83.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the human ftsH protein 16.83 of the 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 is The amino acid may or may not be encoded by the genetic codon; or ( ⁇ ) a type in which one of one or more amino acid residues A group is substituted by another group to include a substituent; or (in.) Such that 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 Species, in which the additional amino acid sequence is fused into a mature polypeptide, such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a proteinogen sequence, as described herein, such fragments, derivatives and analogs It is 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 a SBQ ID NO: 2 amino acid sequence.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a full-length polynucleotide sequence of 2249 bases, and its open reading frame 521-9 9 2 encodes 153 amino acids.
  • this polypeptide has a similar expression profile with human f t sH protein, and it can be deduced that the human f t sH protein 1 6.83 has similar functions as human f t s H protein.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DM forms include cDNA, genomic DNA, or synthetic DM.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SBQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide 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) Used when crossing Denaturing agents, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% F i co ll, 42 ° C, etc .; or (3) the same only between the two sequences Crosses occur only when the sex is at least 95%, and more preferably 97%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 cores Glycylic acid or more.
  • Nucleic acid fragments can also be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding human ftsH protein 16.83.
  • 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 f t sH protein 16.83 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA 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.
  • Various methods have been used to extract mRNA, and kits are also commercially available (Qiagene).
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecule Clinging, A Laboratory Manua, Cold Spring Harbor Labora tory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When combined with polymerase reaction technology, even very small expression products can be cloned.
  • genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DM or DNA-RNA hybridization; (2) the presence or absence of a marker gene function; (3) determination of the level of human ft sH protein 16.83 transcripts; ( 4) Detecting gene-expressed protein products by immunological techniques or 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. In addition, 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).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect protein products expressed by the human ftsH protein 16.83 gene expression.
  • a method using DNA technology to amplify DNA / MA (Saiki, et al. Science 1985; 230: 1350-1354) 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 Selected and synthesized by conventional methods.
  • the amplified DM / RNA fragment can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be 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 the vector of the present invention or directly using a human ftsH protein 16.83 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology.
  • a polynucleotide sequence encoding the human ftsH protein 16.83 may be inserted into a vector to form a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (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.
  • 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 at a later stage of the origin of replication, polyoma enhancers and adenovirus enhancers at the late side of the origin of replication. '
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human f t sH protein 16.83 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.
  • Escherichia coli, Streptomyces bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells insect cells such as fly S 2 or Sf 9
  • animal cells such as CH0, COS or Bowes melanoma cells, etc. .
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DM can be harvested after exponential growth and treated with CaC ⁇ .
  • the steps used are well known in the art.
  • the alternative is to use MgC l 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 liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human f t sH protein 16.
  • 83 (Scence, 1984; 224: 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. When the host cell grows to After 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 isolated 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 chromat
  • FIG. 1 is a comparison diagram of gene chip expression profiles of the inventor's f t sH protein 16. 83 and human f tsH protein.
  • the upper graph is a graph of the expression profile of human f t sH protein 16.
  • 83, and the lower graph is the graph of the expression profile of human f t sH protein.
  • 1-bladder mucosa 2- PMA + Ecv304 cell line, 3- LPS + Ecv304 cell line thymus, 4-normal fibroblasts 1024NC, 5-Fibroblas t, growth factor stimulation, 10 li, 6-scar growth into fc Factor stimulation, 1013HT, 7-scar into fc without stimulation with growth factor, 1013H (:, 8-bladder cancer construct cell W, 9-bladder cancer, 10-bladder cancer, 1 liver cancer, 12-liver cancer cell line, 13-fetal skin, 14-spleen, 15-prostate cancer, 16-jejunum adenocarcinoma, and cardia cancer.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated human f tsH protein 16.83. 17kDa is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention
  • the determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0774B10 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • the results show that the 0774B10 clone contains a full-length cDNA of 2249bp (as shown in Seq ID NO: 1), and has a 461bp open reading frame (0RF) from 521bp to 982bp, encoding a new protein (such as Seq ID NO : Shown in 2).
  • This clone pBS-0774B10 and the encoded protein was named human ftsH protein 16.83.
  • Example 2 Cloning of the gene encoding human ftsH protein 16.83 by RT-PCR
  • CDNA was synthesized using fetal brain cell total RNA as a template and oligo-dT as a primer for reverse transcription reaction. After purification using Qiagene's kit, the following primers were used for PCR amplification:
  • Primerl 5'- GAGATATGTGAGAACTCTGTACTT -3, (SEQ ID NO: 3)
  • Primer2 5,-GTAAAGCAGACATGACAGTTTATT-3, (SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp at the 5 'end of SEQ ID NO: 1;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Conditions for the amplification reaction 50 ⁇ l of reaction volume containing 50 ol / L C1, 10mniol / L Tris-Cl, (pH8.5), 1.5 ⁇ l / L MgCl 2 , 200 ⁇ mol / L L dNTP, lOpmol primer, 1U Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min.
  • ⁇ -actin was set as a positive control and template blank was set as a negative control.
  • the amplified product was purified using a QIAGEN kit, and ligated to a pCR vector (Invitrogen product) using a TA cloning kit.
  • the DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1 to 2249bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human ftsH protein 16.83 gene expression:
  • Electrophoresis was performed on a 1.2% agarose gel containing 2 g of RNA on 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 mM EDTA-2.2M formaldehyde. It was then transferred to a nitrocellulose membrane. Preparation cc- 32 P dATP with 32 P- DNA probe labeled by the random primer method. The DM probe used was the 16.83 coding region sequence (521bp to 982bp) of the PCR amplified human ftsH protein shown in FIG. 1.
  • 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 ° /.
  • the filter was washed in 1 x SSC-0. / SDS at 55 n C for 30 min. Then, it was analyzed and quantified using a Phosphor Imager.
  • Example 4 In vitro expression, isolation and purification of recombinant human ftsH protein 16.83
  • Primer3 5 a CCCCATATGATGCTCTTCTTCCAGATGCCTCAC- 3, (Seq ID No: 5)
  • Priraer4 5'-CATGGATCCCTATTCAAGAAGCCCAAGATAGAA-3 '(Seq ID No: 6)
  • the 5' ends of these two primers contain Ndel and BamHI restriction sites, respectively.
  • the coding sequences for the 5 'and 3' ends of the gene of interest are followed, respectively.
  • the Ndel and BamHI restriction sites correspond to the selectivity within the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Digestion site.
  • PCR was performed using the PBS-0774B10 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: 10 pg of pBS-0774B10-containing plasmid in a total volume of 50 ⁇ l, primers Primer-3 and Primer-4 were lpmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1, respectively. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles. Nde I and BaniH I were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligation product was transformed into E. coli DH5CC using the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 30 g / ml), positive clones were screened by colony PCR method and sequenced. A positive clone (PET-0774B10) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (a product of Novagen) by the calcium chloride method.
  • the host bacteria BL21 (pET-0774B10) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1 leg oi / L, continue to cultivate for 5 hours.
  • the bacteria were collected by centrifugation, and the supernatant was collected by ultrasonication. The supernatant was collected by centrifugation.
  • Example 5 Production of anti-human ftsH protein 16.83 antibody
  • the following peptides specific to human ft sH protein 16.83 were synthesized using a peptide synthesizer (product of PE company): NH2-Met-Leu-Phe-Phe-G ln-Met-Pro-H i sI l ⁇ -Tyr-Leu -Ser-G 1 u- Asn-Cy s- COO H (SEQ ID NO: 7).
  • the peptide was coupled with hemocyanin and bovine serum albumin to form a complex. For the method, see: Avrameas, et al.
  • 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 example 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 to hybridize the fixed polynucleotide sample to the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing 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 uses higher intensity membrane washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention;
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the spot imprint 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 fragment selected from the polynucleotide SEQ ID NO: 1 of the present invention for use as a hybridization probe shall be Following the following principles and several aspects to consider:
  • the preferred range of probe size is 18-50 nucleotides
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, then the primary probe should not be used;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutant sequence of the gene fragment of SEQ ID NO: 1 or its complementary fragment (41Nt):
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • Two NC membranes are required for each probe for subsequent experiments.
  • the film is washed with high-strength conditions and strength conditions, respectively.
  • the sample membrane was placed in a plastic bag, and 3-1 (kg of pre-hybridization solution (lOxDenhardt's; 6xSSC, 0.1 mg / ral CT DNA (calf thymus DNA).) was added. After the bag was sealed, it was 68 ° C in water. Shake for 2 hours.
  • 3-1 kg of pre-hybridization solution (lOxDenhardt's; 6xSSC, 0.1 mg / ral CT DNA (calf thymus DNA).
  • the hybridization experiments performed under low-intensity membrane washing conditions did not differ significantly in the radioactivity of the hybridization spots of the above two probes; while the hybridization experiments conducted under high-intensity membrane washing conditions, the radioactive intensity of hybridization spots of probe 1 was significantly stronger than The radioactive intensity of the hybridization spot of the other probe. Therefore, the presence and differential expression of the polynucleotide of the present invention in different tissues can be analyzed qualitatively and quantitatively with the probe 1.
  • Gene microarray or DNA microarray is a new technology 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 slopes. And silicon, and then use fluorescence detection and computer software to compare and analyze the data, so as to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as a target DM for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially diseases related genes such as tumors; diagnosis of diseases such as heredity disease.
  • the specific method steps have been reported in the literature, for example, please refer to the literature DeRi Si, L L., Lyer, V. & Brown, P. 0.
  • 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 amplified by PCR respectively. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 ⁇ . The spotted slides were hydrated, dried, and cross-linked in a UV cross-linker. After elution, the slides were fixed to fix the DNA on the glass slides to prepare chips. The specific method steps have been variously reported in the literature. The post-spot processing steps of this embodiment are:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and mRNA was purified using Oligotex mRNA Midi Kit (purchased from QiaGen).
  • the fluorescent test J Cy3dUTP (5-Amino-propargy l-2'-deoxyur idine 5 -tr ip ate coupled to Cy3 fluorescent dye, purchased from Araersham Phamacia Biotech) was used to label the mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP (5-Amino-propargyl-2'-deoxyur idine 5'-triphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech) to label the specific tissue (or stimulated cell line) raRNA of the body, and prepare the probe after purification .
  • Cy3dUTP 5-Amino-propargy l-2'-deoxyur idine 5 -tr ip ate coupled to Cy3 fluorescent dye, purchased from Araers
  • the probes from the two types of tissues and the chip were hybridized in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, washed with a washing solution (lx SSC, 0.2 SDS) at room temperature, and then scanned with a ScanArray 3000 scanner. (Purchased from General Scanning, USA) for scanning. The scanned images were analyzed and processed with Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are bladder mucosa, PMA + Ecv304 cell line, LPS + Ecv304 cell line thymus, normal fibroblasts 1024NC, Fibroblast, growth factor stimulation, 1024NT, scar-like fc growth factor , 1013HT, scar into fc without stimulation with growth factors, 1013HC, bladder cancer cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunal adenocarcinoma, cardia cancer. Based on these Cy3 / Cy5 ratios, a bar graph is drawn. (figure 1 ) . It can be seen from the figure that the expression profile of human ftsH protein 16.83 and human ftsH protein according to the present invention are very similar. Industrial applicability
  • polypeptides of the present invention as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.
  • Cell division and growth are an important regulatory process for the growth and reproduction of organisms.
  • the mutation or abnormal expression of this protein will directly affect the division and proliferation of related cells in the body, and then cause various related diseases.
  • the protein is usually closely related to the development of some developmental disorders, related tissue tumors and cancers in the body.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of human ft sH protein, and both have similar biological functions.
  • the polypeptide of the present invention plays an important regulatory role in various cell cycle processes in vivo, and regulates the division and proliferation of related cells.
  • the mutation or abnormal expression of this protein will directly affect the division and proliferation of related cells in the body, and then lead to the development of embryo malformations and tumor diseases. These diseases include but are not limited to:
  • Cleft lip (most common, with alveolar cleft and cleft palate), cleft palate, oblique cleft face, cervix, cervix, etc .;
  • Horizontal absence congenital short limbs: no arms, no forearms, no hands, no fingers, no legs, no toes, etc .; longitudinal absences: radial / ulnar abscess of upper extremity, tibia / fibula absent of lower extremity, etc .;
  • Limb differentiation disorder Absence of a certain muscle or muscle group, joint dysplasia, bone deformity, bone fusion, multi-finger (toe) deformity, numb (toe) deformity, horse tellurium varus etc .;
  • Thyroglossal duct cyst Thyroglossal duct cyst, gastrointestinal atresia or stenosis, ileal diverticulum, umbilical diaphragm, congenital umbilical hernia, congenital nonganglionic megacolon, impervious anus, abnormal bowel transition, bile duct atresia, circular pancreas, etc .;
  • Atrial septal defect aorta and lung Arterial dislocation, aortic or pulmonary stenosis), open arterial ducts, etc .
  • ventricular septal defect abnormal separation of arterial trunk (aorta and lung Arterial dislocation, aortic or pulmonary stenosis), open arterial ducts, etc .;
  • neural tube defects no cerebral malformations, spina bifida, spinal meningocele, hydrocephalous meningoencephalocele), hydrocephalus inside / outside the brain, etc .
  • Papilloma squamous cell carcinoma [skin, nasopharynx, larynx, cervix], adenoma (carcinoma) [breast, thyroid], mucinous / serous cystadenomas (carcinoma) [ovary], basal cell carcinoma [head and face Skin], (malignant) polytype adenoma [extending gland], papilloma, transitional epithelial cancer [bladder, renal pelvis], etc .; 2.
  • Mesenchymal tissue :
  • lymphoid hematopoietic tissue Third, lymphoid hematopoietic tissue:
  • Malignant lymphoma neck, mediastinum, mesenteric and retroperitoneal lymph nodes], various leukemias [lymphoid hematopoietic tissue], multiple myeloma [push / thoracic / rib / skull and long bone], etc .;
  • Nerve fiber [systemic cutaneous nerve / deep nerve and internal organs], (malignant) schwannoma [nervous of head, neck, limbs, etc.], (malignant) glioblastoma [brain], medulloblastoma [ Cerebellum], (malignant) meningiomas [meninges], ganglioblastoma / neuroblastoma [mediastinum and retroperitoneum / adrenal medulla], etc .;
  • malignant melanoma skin, mucous membrane
  • (malignant) hydatidiform mole chorionic epithelial cancer [uterine]
  • (malignant) supporter cells stromal cell tumor
  • (malignant) granulosa cell tumor ovarian, testicular] fine Blastoma [testis], asexual cell tumor [ovary], embryonal cancer [testis, ovary], (malignant) teratoma [ovary, testis, mediastinum and palate tail], etc .
  • malignant melanoma skin, mucous membrane
  • hydatidiform mole chorionic epithelial cancer [uterine]
  • (malignant) supporter cells stromal cell tumor
  • (malignant) granulosa cell tumor ovarian, testicular] fine Blastoma [testis]
  • asexual cell tumor ovary
  • embryonal cancer testis, ovary
  • (malignant) teratoma
  • polypeptide of the present invention and the antagonist, agonist and inhibitor of the polypeptide can be directly used in various diseases Treatment, especially embryo developmental abnormalities, tumor diseases, etc.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human sH protein 16.83.
  • Agonists increase human ftsH protein 16.83 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 ftsH protein 16.83 can be cultured with labeled human ftsH protein 16.83 in the presence of drugs. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human ftsH protein 16.83 include screened antibodies, compounds, receptor deletions and analogs. Antagonist of human f tsH protein 16.83 can bind to human f tsH protein 16.83 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions.
  • human ftsH protein 16.83 When screening compounds as antagonists, human ftsH protein 16.83 can be added to the bioanalytical assay to determine whether the compound is an antagonist by measuring the effect of the compound on the interaction between human ftsH protein 16.83 and its receptor. 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 ftsH protein 16.83 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, generally 16.83 molecules of human ftsH protein should be labeled.
  • the present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against the 16.83 epitope of human ftsH protein. 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 direct injection of human ftsH protein 16.83 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 adjuvant.
  • Techniques for preparing monoclonal antibodies against human ftsH protein 16.83 include, but are not limited to, hybridoma technology (ohler and Mil stein. Nature, 1975, 256: 495-497), triple tumor technology, human B-cell hybridoma technology, EBV-hybridization Tumor technology, etc.
  • Chimeric antibodies that bind human constant regions to non-human variable regions can be produced using conventional techniques (Morrison et al, PNAS, 1985, 81: 6851). 0 Other techniques for producing single-chain antibodies (US Pat No. 4946778) can also be used to produce single chain antibodies against human ftsH protein 16.83
  • Antibodies against human ftsH protein 16.83 can be used in immunohistochemical techniques to detect human ftsH protein 16.83 in biopsy specimens.
  • Monoclonal antibodies that bind to human ftsH protein 16.83 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 It depends on the location of tumor cells and whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • human ftsH protein 16.83 high affinity monoclonal antibody can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human ftsH protein 16.83-positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human ftsH protein 16.83.
  • Administration of an appropriate dose of antibody can stimulate or block the production or activity of human ftsH protein 16.83.
  • the invention also relates to a diagnostic test method for the quantitative and localized detection of 16.83 levels of human ftsH protein.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human f tsH protein 16.83 detected in the test can be used to explain the importance of human f tsH protein 16.83 in various diseases and to diagnose diseases in which human f tsH protein 16.83 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.
  • the polynucleotide encoding human ftsH protein 16.83 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 ftsH protein 16.83.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human ftsH protein 16.83 to inhibit endogenous human ftsH protein 16. S3 activity.
  • a variant human ftsH protein 16.83 may be a shortened human ftsH protein 16.83 that lacks a signaling domain, although it can bind to downstream substrates, but lacks signaling activity.
  • recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or 'activity of human ftsH protein 16.83.
  • Expression vectors derived from viruses such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, and parvovirus can be used to transfer a polynucleotide encoding the human ftsH protein 16.83 into cells.
  • viruses such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, and parvovirus
  • viruses such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, and parvovirus
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding the human ftsH protein 16.83 can be found in existing literature (Sambrook, et ai.).
  • 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 that inhibit human ftsH protein at 16.83 mRM are also within the scope of the present invention.
  • Ribozymes are enzyme-like RNA molecules that can specifically break down specific RNAs. The mechanism of action is A ribozyme molecule specifically hybridizes to a complementary target RNA for endonucleation.
  • DNA and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as solid-phase phosphate amide chemical synthesis to synthesize oligonucleotides.
  • Antisense RM molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA.
  • This DM sequence has been integrated downstream of the RNA polymerase promoter of the vector.
  • it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside linkages should use phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human ftsH protein 16.83 can be used for the diagnosis of diseases related to human ftsH protein 16.83.
  • a polynucleotide encoding human ftsH protein 16.83 can be used to detect the expression of human ftsH protein 16.83 or the abnormal expression of human ft sH protein 16.83 in a disease state.
  • a DNA sequence encoding human ftsH protein 16.83 can be used to hybridize biopsy specimens to determine the expression of human ftsH protein 16.83.
  • Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. These techniques and methods are publicly available mature technologies, and related kits are commercially available.
  • a part or all of the polynucleotide of the present invention can be fixed as a probe on a microarray or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Human ftsH protein 16.83 specific primers for MA-polymerase chain reaction (RT-PCR) amplification in vitro can also detect the human ftsH protein ⁇ 6.83 transcription products.
  • Detection of mutations in the human ftsH protein 16.83 gene can also be used to diagnose human ftsH protein 16.83-related diseases.
  • Human ftsH protein 16.83 mutant forms include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human ft sH protein 16.83 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 the expression of proteins, so Northern blotting and Western blotting can be used to indirectly determine the presence or absence of mutations in a gene.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for marking chromosome positions.
  • its' an important first step is to locate these DM 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 by a similar method, 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, pre-screening of chromosomes using labeled flow sorting, and pre-selection of hybridization, thereby constructing a chromosome-specific cDNA library.
  • 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, Mendel ian Inheritance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been 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 with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients that 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 containing one or more ingredients of the pharmaceutical composition of the present invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which reminders permit their administration on the human body by government agencies that manufacture, use, or sell them.
  • the polypeptide of the present 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 ftsH protein 16.83 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human ftsH protein 16.83 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine humaine 16.83 ftsH, et un polynucléotide codant ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment de malformations lors du développement de l'embryon et de tumeurs. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant la protéine humaine 16.83 ftsH.
PCT/CN2001/000946 2000-06-14 2001-06-11 Nouveau polypeptide, proteine humaine 16.83 ftsh, et polynucleotide codant ce polypeptide WO2002000829A2 (fr)

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Title
DATABASE GENBANK [Online] 06 March 2000 COTTRELL, M.T. ET AL. Retrieved from NCBI, accession no. GI:6646914 Database accession no. (AAF21107.1) *
DATABASE GENBANK [Online] 23 November 1999 BIRD, C. ET AL. Retrieved from NCBI, accession no. GI:3080381 Database accession no. (AL022578.1) *

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AU8953401A (en) 2002-01-08
WO2002000829A3 (fr) 2002-05-02
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