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WO2001074888A1 - Nouveau polypeptide, $g(b)-preprotachykinine humaine 9, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, $g(b)-preprotachykinine humaine 9, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001074888A1
WO2001074888A1 PCT/CN2001/000188 CN0100188W WO0174888A1 WO 2001074888 A1 WO2001074888 A1 WO 2001074888A1 CN 0100188 W CN0100188 W CN 0100188W WO 0174888 A1 WO0174888 A1 WO 0174888A1
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Prior art keywords
polypeptide
polynucleotide
protakinin
human beta
sequence
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PCT/CN2001/000188
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc
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Shanghai Biowindow Gene Development Inc
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Priority to AU44039/01A priority Critical patent/AU4403901A/en
Publication of WO2001074888A1 publication Critical patent/WO2001074888A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/22—Tachykinins, e.g. Eledoisins, Substance P; Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, namely human beta-protakinin 9, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique
  • Tachykinins are a group of small peptides that exist in the brain and peripheral tissues. They play an important role as neurotransmitters and hormones. Tachykinin has a ubiquitous C-terminal amino acid sequence as a characteristic marker, namely -Phe-X-Gly- Leu-Met- NH. In the second subunit, where X is a hydrophobic or aromatic residue [Erspamer, V., Trends in Neuroscience, 4, 267—'269, 1981]. The first tachykinin discovered was substance P, which is involved in the transmission of pain stimuli in the spinal cord, and some substances are released from the sensory nerves of the skin and play a role in the inflammatory response.
  • tachykinins such as neurokinins A and B [Kimura, S., Oada, M., Sugita, ⁇ ., Kanazawa, I. and Munekata, E., Proc Jap. Acad. Series B , 59, 101-104, 1983], and the larger tachykinin in the central nervous system, neuropeptide K [Tatemoto, K., Lundberg, JM, Jornvall, H and Mutt, V., Biochem. Biophys. Res Comm., 128, 947-953, 1985].
  • Substance P is derived from two larger peptide precursors, ⁇ - and P-protachykinin (PPT).
  • the mRNA encoding ex- and ⁇ -protakinin is derived from a single PPT gene, and then the tissue-specific RNA is sheared to generate ⁇ -, ⁇ -protakinin.
  • Substance P and neurokinin A function through different tachykinin receptors, which are distributed in different locations in the nervous system.
  • ⁇ -protachykinin is the common precursor of neurokinin A, P and neuropeptide K.
  • the human ⁇ -protakinin original chain contains regions encoding substance P and neurokinin A, and each region is flanked by basic amino acid residues. The sequence containing these basic amino acid residues is the site of post-translational processing. point.
  • a sequence of more than thirty residues near the C-terminus of human ⁇ -PPT encodes a neuropeptide K, which is an N-terminal extension of neurokinin A [Harmar AJ, Armstrong A, Pascal 1 JC, Chapman K, etc ., FEBS Lett 1986 Nov 10; 208 (1): 67- 72].
  • the cleavage site of human ⁇ -PPT signal sequence is between 19-alanine and 20-glutamic acid. After cleavage, 110 amino 'length prohormones will be produced. A series of hormone peptides.
  • Some people's carcinoid tumors are caused by the secretion of peptides derived from ⁇ -protakinin.
  • human laryngeal carcinoid tumors contain high concentrations of immunoreactive substance P. This tumor causes patients to secrete saliva. And local pain, so ⁇ -protakinin and its antagonists, Inhibitors and agonists can be used to diagnose and prevent certain carcinoid tumor diseases.
  • human beta-protakinin 9 protein plays an important role in important functions of the body such as copper node cell division and embryo development, and it is believed that a large number of proteins are involved in these regulatory processes, so the identification of more proteins has been required in the art. Many human beta-protakinin 9 proteins are involved in these processes, especially the amino acid sequence of this protein is identified. Isolation of the newcomer beta-pronto 3 ⁇ 4peptide 9 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 diagnosis and / or treatment of a disease, so it is important to isolate its coding DNA. Disclosure of invention
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding human beta-protakinin 9.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding human beta-protakinin 9. '
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention-human beta-protakinin 9.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors against human beta-protachykinin 9 of the polypeptide of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating a disease associated with abnormality of human beta-protakinin-9.
  • 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 present invention also relates to an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: Its variant:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) having SEQ ID D 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; and a culture cell
  • 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
  • a culture cell The method for preparing a polypeptide of the present invention by describing a host cell and recovering an expressed product is described.
  • 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 beta-protakinin 9 protein, which comprises utilizing a polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the present invention also relates to a method for in vitro detection of a disease or susceptibility to disease associated with abnormal expression of human be t a-protakinin 9 protein, which comprises detecting a polypeptide in a biological sample or a coding polynucleotide sequence thereof. Mutates, or detects 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 preparation of the polypeptide and / or polynucleotide of the present invention for the treatment of laryngeal carcinoma tumors, neurological diseases, endocrine diseases, immune diseases, inflammation, various tumors, growth and development disorders, blood diseases, Use of HIV infection or other drugs due to abnormal expression of human beta-protakinin-9.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to a peptide, 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
  • polypeptide or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a protein or polynucleotide “variant” refers to an amino acid sequence having one or more amino acids or nucleotide changes, or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants may have "conservative" changes in which the substituted amino acid 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 in appropriate animals or cells and to bind to specific antibodies.
  • An "agonist” refers to a molecule that can cause changes in the protein to regulate the activity of the protein when combined with human beta-protakinin.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind human beta-protakinin 9.
  • Antagonist refers to a biological activity or immunity that can block or modulate human be t a-protakinin 9 when combined with human be t a-protakinin 9 Chemically active molecules. Antagonists and inhibitors can include proteins, nucleic acids, carbohydrates or any other molecule that can bind human beta-protakinin 9.
  • Regular refers to a change in the function of human be t a-protakinin 9, including an increase or decrease in protein activity, a change in binding properties, and any other organism of human be t a-protakinin 9 Changes in nature, function, or immunity.
  • substantially pure ' means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify human beta-protakinin by standard protein purification techniques 9. Basically pure human be ta-protakinin 9 can generate a single main band on a non-reducing polyacrylamide gel. The purity of human be ta-protakinin 9 polypeptide can be analyzed by amino acid sequence ... '''
  • Complementary refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence C-T-G-A
  • complementary sequence G-A-C-T.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology A partially complementary sequence that at least partially inhibits the hybridization of a fully complementary sequence to a target nucleic acid. The inhibition of such hybridization can be detected by performing hybridization (Southern blotting or Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. It does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences.
  • the percentage identity can be determined electronically, such as by the MEGALIGN program (Lasergene software package, DNASTAR, Inc., Madison Wis.).
  • the MEGALIGN program can compare two or more sequences according to different methods, such as the Cluster method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). 0
  • the Cluster method arranges groups of sequences by checking the distance between all pairs. Into clusters. 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: The number of matching residues between sequence A and sequence X 100 The number of residues in sequence A-the number of spacer residues in sequence A Number of interval residues in a sequence B
  • the percent identity between nucleic acid sequences can also be determined by the Cluster method or by methods known in the art such as Jotun Hein (Hein J., (1990) Methods in emzumology 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 a “sense strand.”
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. Such a chemical modification may be an alkyl, acyl or amino group in which a hydrogen atom is replaced.
  • a nucleic acid derivative may encode a polypeptide that retains the main biological properties of a natural molecule.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and? ⁇ It can specifically bind to human beta-protakinin 9 epitopes.
  • Humanized antibody means that the amino acid sequence of a non-antigen-binding region is replaced with a human antibody Antibodies that are similar but still retain the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not part of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances in the natural state .
  • isolated human beta-prokininogen 9 refers to human beta-prokininogen 9 which 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 beta-protakinin 9 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human beta-protakinin 9 peptides can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide: "A human beta-protakinin 9 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 or a natural polypeptide Synthetic polypeptide, preferably recombinant polypeptide.
  • the polypeptide of the present invention can be a naturally purified product or a chemically synthesized product, or can be obtained from a prokaryotic or eukaryotic host (for example, bacteria, yeast, higher plants, insects and mammals) using recombinant technology. Cell).
  • the polypeptide of the invention may be glycosylated or may be non-glycosylated.
  • the polypeptide of the invention may also or may not include the initial methionine residue Base.
  • the invention also includes fragments, derivatives and analogs of human beta-protakinin 9.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the human beta-protakinin 9 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) such a type in which one or more amino acid residues are substituted with other groups to include a substituent; or (III) such A type in which a mature polypeptide is fused with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or a UV type) in which an additional amino acid sequence is fused into a mature polypeptide and the polypeptide sequence is formed Columns (such as leader sequences or secreted sequences or sequences used to purify this polypeptide or protease sequences) As set forth herein, such fragments, 00 derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a full-length polynucleotide sequence of 1762 bases, and its open reading frames 93-353 encode 86 amino acids.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 in the present invention, but which differs from the coding region sequence shown in SEQ ID NO: 1.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various plus 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, which may be one or more nucleosides: substitutions, deletions, or insertions of acids, but without substantially altering the polypeptides they encode.
  • 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) added during hybridization Use a denaturant, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc .; or (3) the identity between the two sequences is at least 95% Above, more preferably 97% or more hybridization occurs. and the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 cores. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding human beta-protakinin 9.
  • 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 human beta-protakinin 9 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 DM sequences is often the method of choice. The more commonly used method is the separation of cDM sequences.
  • the standard method for isolating the cDM 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. There are many mature techniques for extracting mRNA, and kits are also commercially available (Qiagene).
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When combined with polymerase reaction technology, even very few 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): (1) DNA-DNA or DM-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of the level of human beta-pretakinin 9 transcripts (4) Detecting protein products expressed by genes through 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.
  • the length of the probe is usually within 2000 nucleotides, and preferably within 1000 nucleotides.
  • the probe used here is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product for detecting the expression of human beta-protakinin 9 gene Epidemiological techniques include Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • the method of using PCR to amplify DNA / RNA is preferred to obtain the gene of the present invention: Especially when it is difficult to obtain full-length CDM from the library, It is preferable to use the RACE method (RACE-rapid cDNA end rapid amplification method).
  • Primers used for PCR can be appropriately selected according to the polynucleotide sequence information of the present invention disclosed herein, and can be synthesized by 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 determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequencing can also be performed using a 'commercial sequencing kit' 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 that is genetically engineered using the vector of the present invention or directly using a human beta-protakinin 9 coding sequence, and that the present invention is produced by recombinant technology Polypeptide method.
  • a polynucleotide sequence encoding human beta-protakinin 9 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 expressed in bacteria (Rosenberg, et al.
  • any plasmid and vector can be used to construct recombinant expression vectors.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • An expression vector for DNA sequences and appropriate transcriptional / translational regulatory elements include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to the i
  • 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. Examples include SV40 enhancer of 100 to 2 70 base pairs on the late side of the origin of replication, polyoma enhancer and adenovirus enhancer on the late side of the origin of replication, and the like.
  • 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 beta-protakinin 9 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetic engineering containing the polynucleotide or the recombinant vector.
  • Host cell refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf 9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCli.
  • 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.
  • polynucleotide sequence of the present invention can be used to express or produce recombinant human beta-protakinin 9 (Scence, 1984; 224: 1431). Generally, the following steps are taken:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. When host cells grow to proper After inducing the cell density, the appropriate promoter (such as temperature conversion or chemical induction) is used to induce the selected promoter, and the cells are cultured for a period of time.
  • the appropriate promoter 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 include, but are not limited to: conventional renaturation treatment, protein precipitation: 'detergent treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid layer techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitation: 'detergent treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC)
  • FIG. 1 is a comparison diagram of gene chip expression profiles of the inventors beta-protakinin 9 and human beta-protakinin.
  • the upper graph is a graph of the expression profile of human beta-protakinin 9.
  • the lower graph is the graph of the expression profile of human beta-protachykinin.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of human beta-protakinin 9 isolated.
  • 9KDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • RNA Human fetal brain total RNA was extracted by one step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using the Quik mRNA Isolation Kit (Qiegene). 2ug poly (A) mRNA forms cDM by reverse transcription.
  • the Smart cDNA cloning kit purchased from Clontech was used to insert the cDNA fragment into the multiple cloning site of pBSK (+) vector (Clontech) to transform DH5 ⁇ , and the bacteria formed a cDM 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 DNA sequence database (Genebank), and one of the clones was found.
  • the 0400g09 cDNA sequence is 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 0400g09 clone contains a full-length cDNA of 1762bp (as shown in Seq ID NO: 1), and has a 261bp open reading frame (0RF) from 93bp to 353bp, encoding a new protein (such as Seq ID NO : Shown in 2).
  • Example 2 Cloning of a gene encoding human beta-protakinin 9 by RT-PCR
  • CDNA was synthesized using fetal brain 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 — CAGCTACTATAAACATTTGTGCAC —3, (SEQ ID NO: 3)
  • Primer2 5'- ATTTGAAAGAAACAAAATTTTATT-3, (SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp of the 5th end of SEQ ID NO: 1;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification conditions 50 ⁇ l of KC1, 10 ⁇ l / L of Tris-Cl, ( ⁇ 8.5), 1.5 mmol / L MgCl 2 , 200 ⁇ / L dNTP, l'Opmol primer, 1U of Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94. C 30sec; 55. C 30sec; 72 ° C 2min 0 During RT-PCR, ⁇ -act in was set as the positive control and template blank was used as the 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 1762bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human beta-protakinin 9 gene expression:
  • RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] 0
  • This method involves acid guanidinium thiocyanate-chloroform extraction. That is, the tissue is homogenized with 4M guanidinium isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1) are added. ) And centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • RNA probes were the PCR amplified human beta-protakinin 9 coding region sequence (93bp to 353bp) shown in FIG. 1.
  • a 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 (pH7.4) -5 x SSC-5 ⁇ Denhardt's solution and 200 ⁇ g / ml salmon sperm DNA. 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 beta-protakinin 9
  • Primer3 5'- CATGCTAGCATGTGTAAGCACGTGTTTAACTTA -3 '(Seq ID No: 5)
  • Primer4 5'- CATGGATCCTTAAAATGGACAAAAGATTTGAAC -3, (Seq ID No: 6)
  • the 5' ends of these two primers contain Nhel and BamHI restriction sites, respectively. : 'The following are the coding sequences of the 5' and 3 'ends of the gene of interest, respectively.
  • the Nhel and BamHI restriction sites correspond to the selection on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Sex endonuclease site.
  • the PCR reaction was performed using pBS-0400g09 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-0400g09 plasmid, primers 1 ⁇ 11161 ⁇ -3 and ⁇ 1 ⁇ 11161 "-4 points and 1] is 1 ( ⁇ [1101, Advantage polymerase Mix (Clontech) Product) 1 ⁇ 1.
  • Cycle parameters 94 ° C 20s, 60 ° C 30s, 68. C 2 rain, 25 cycles in total. Nhel and BamHI were used to double-enzyme the amplification product and plasmid pET-28 (+), respectively.
  • the ligation product was transformed into Escherichia coli DH5 ⁇ by the calcium chloride method, cultured overnight on LB plates containing kanamycin (final concentration 30 g / ml), and then colonies were used.
  • the positive clones were screened by PCR and sequenced.
  • the positive clones (pET-0400g09) with the correct sequence were selected.
  • the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method.
  • the following peptides specific to human beta-protakinin 9 were synthesized using a peptide synthesizer (product of PE): NH2-Met-Cys-Lys-His-Val-Phe-Asn-Leu-Glu-Gly-Gly- Leu-Gln-Thr-Leu-C00H (SEQ ID NO: 7).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method.
  • Acid sequence or homologous polynucleotide sequence.
  • Filter hybridization methods include dot blotting, Southern imprinting, Nor thern blotting, and copying methods. They all use the same steps to fix the polynucleotide sample to be tested on the filter and then hybridize.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature) 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 second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • oligonucleotide fragments 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
  • 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):
  • C Use an electric homogenizer to homogenize the tissue suspension at full speed until the tissue is completely broken.
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membrane nitrocellulose membrane
  • the 32 P-Probe (the second peak is free ⁇ - 32P -dATP) is prepared.
  • 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 large numbers of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • the specific method steps have been reported in the literature. For example, see the documents DeRisi, JL, Lyer, V. & Brown, PO (1997) Science 278, 680-686. And the documents Helle, RA, Schema, M., Chai, A., Shalom, D., (1997) PNAS 94: 2150-2155.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were 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 spotting instrument (purchased from Cartesian, USA). The distance is 280 ⁇ m. The spotted slides were hydrated, dried, and cross-linked in a UV cross-linking instrument. After elution, the DNA was fixed on the glass slide to prepare a chip. The specific method steps have been variously reported in the literature. The post-spotting processing steps of this embodiment are:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) using a one-step method, and the mRNA was purified using Oligotex mRNA Midi Kit (purchased from QiaGen).
  • the fluorescent reagent Cy3dUTP 5— Amino— propargy 2'— deoxyuridine 5'— triphate coupled to Cy3 fluorescent dye (purchased from Amersham Pharaacia Biotech) was used to label mRNA of human mixed tissues, and the fluorescent reagent Cy5dUTP (5- Amino-propargy 2'-deoxyuridine 5'-tr iphate coupled to Cy5 fluorescent, dye, purchased from Amersham Phamac ia Biotech) labeled the mRNA of a specific tissue (or stimulated cell line) of the body, and purified the probe to prepare a probe.
  • Cy3dUTP 5— Amino— propargy 2'— deoxyuridine 5'— triphate coupled to Cy3 fluorescent dye (purchased from Am
  • 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 (1 x SSC, 0.2% SDS) at room temperature, and then scanned with ScanArray 3000.
  • the scanner purchased from General Scanning Company, USA
  • the scanned image was analyzed and processed with Imagene software (Biodiscovery Company, 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, Arsenic stimulated the L02 cell line and prostate tissue for 1 hour. Based on these 13 Cy3 / Cy5 ratios, a bar graph is drawn. (figure 1) . It can be seen from the figure that the expression profile of human beta-protakinin 9 and human beta-protakinin according to the present invention are very similar. Industrial applicability
  • polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infections and immune diseases.
  • Tachykinins are a group of small peptides that exist in the brain and peripheral tissues. They play an important role as neurotransmitters and hormones. ⁇ -protachykinin is a common precursor of neurokinin A, P substance and neuropeptide K. Substance P can be involved in the transmission of pain stimuli in the spinal cord, inflammatory responses, and so on. Neurokinin A, Neuropeptides work through different tachykinin receptors, which are distributed in different locations in the nervous system.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of human ⁇ -protakinin, and both have similar biological functions. It acts as a precursor of substance P in the body, neurokinin A and neuropeptide K Its derivatives can be used as neurotransmitters to participate in a variety of neural function regulation, endocrine regulation, can be used as inflammatory transmitters to participate in inflammatory response, can be used as immune responders to participate in immune response, and so on. Its abnormal expression will cause the above-mentioned tissue system to malfunction, and cause related diseases.
  • human be t a-protakinin 9 of the present invention will produce various diseases, especially laryngeal carcinoid tumors, neurological diseases, endocrine diseases, immune diseases, inflammation, various tumors, Growth and developmental disorders, including but not limited to:
  • Neurological diseases Glioblastoma, Astrocytoma, Ependymal tumor, Neurofibromas, Pituitary adenoma, Intracranial granuloma, Alzheimer's disease, Parkinson's disease, Chorea, Depression, Epilepsy , Migraine, neuromuscular disease, neurocutaneous syndrome, schizophrenia, acute myelitis, spinal cord compression, trigeminal neuralgia, facial nerve palsy, bulbar palsy, sciatica, Guillain-Barre syndrome, neural tube insufficiency, Brain developmental abnormalities, neuronal migration disorders
  • Endocrine diseases precocious puberty, giant disease and acromegaly, pituitary syndrome, hypohypoxia in adults, dwarfism, cortisolism, adrenal insufficiency, adrenal tumors, diabetes, premenstrual stress, Menopause syndrome, ovarian hypoplasia, amenorrhea, hypogonadism, precocious puberty
  • Immune diseases Systemic lupus erythematosus, rheumatoid arthritis, bronchial asthma, urticaria, specific dermatitis, post-infection myocarditis, scleroderma, myasthenia gravis, Guillain-Barre syndrome, common variable immunodeficiency disease , Primary B-lymphocyte immunodeficiency disease, Acquired immunodeficiency syndrome
  • Inflammation chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, cerebrospinal multiple sclerosis, glomerulonephritis, myocarditis, cardiomyopathy, atherosclerosis, gastric ulcer, cervicitis, Various infectious inflammations
  • Tumors of various tissues laryngeal carcinoma, laryngeal cancer, stomach cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, endometrial cancer, colon cancer, thymic tumor, nasopharyngeal cancer, tracheal tumor
  • Growth and development disorders mental retardation, brain development disorders, skin, fat, and muscular dysplasia, bone and joint dysplasia, various metabolic defects, stunting, dwarfism, Cushing's syndrome Sexual retardation
  • Abnormal expression of human beta-protakinin 9 of the present invention will also produce certain hereditary, hematological diseases and the like.
  • polypeptides of the present invention and the antagonists, agonists and inhibitors of the polypeptides can be directly used in the treatment of diseases, for example, they can treat various diseases, especially laryngeal carcinoma tumors, neurological diseases, endocrine diseases, immune diseases, inflammation, Various tumors, disorders of growth and development, certain hereditary, hematological diseases, etc.
  • the invention also provides screening compounds to identify improving (agonist) or repressing (antagonist) human be t a- Method of Pre-Tachykinin 9 Pharmacy Agonists enhance biological functions such as human beta-protakinin 9 to stimulate cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or a membrane preparation expressing human beta-protakinin 9 can be cultured with labeled human beta-protakinin 9 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human beta-protachykinin 9 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human beta-protakinin 9 can bind to human beta-protakinin 9 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide to make the polypeptide Cannot perform biological functions.
  • human beta-protakinin 9 When screening compounds as antagonists, human beta-protakinin 9 can be added to bioanalytical assays by measuring the effect of compounds on the interaction between human beta-protakinin 9 and its receptor Determine if the compound is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same way as for screening compounds described above. Polypeptide molecules capable of binding to human beta-protakinin 9 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, human beta-protakinin 9 molecules should generally be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against human beta-protakinin 9 epitopes. These antibodies include, but are not limited to.-Polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments from Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human beta-protakinin 9 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 'S adjuvant and so on.
  • Techniques for preparing monoclonal antibodies to human beta-protakinin 9 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma technology , EBV-hybridoma technology, etc.
  • 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).
  • the existing technology for producing single chain antibodies U. S. Pat No. 4946778, can also be used to produce single chain antibodies against human beta-protakinin 9.
  • Antibodies to human beta-protakinin 9 can be used in immunohistochemistry to detect human beta-pretakinin 9 in biopsy specimens.
  • Monoclonal antibodies that bind to human beta-protakinin 9 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-traumatic diagnosis The method is used 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 beta-protakinin 9 high affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human beta-protakinin 9 Positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases associated with human beta-protakinin 9.
  • Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human beta-protakinin 9.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human beta-protakinin 9 levels.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the levels of human beta-protakinin 9 detected in the test can be used to explain the importance of human beta-protakinin 9 in various diseases and to diagnose human beta-protakinin 9 A working disease.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • Polynucleotides encoding human beta-protakinin 9 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 beta-protakinin-9. Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant human beta-protakinin 9 to inhibit endogenous human beta-protakinin 9 activity. For example, a variant human beta-protakinin 9 may be shortened and lack human signaling beta-protakinin 9, although it can bind to downstream substrates, but lacks signaling. active.
  • 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 beta-protakinin-9.
  • Recombinant gene therapy vectors such as viral vectors
  • a variant human beta-protakinin 9 may be shortened and lack human signaling beta-protakinin 9, although
  • the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human beta-protakinin 9.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding human beta-protakinin 9 into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding human beta-protakinin 9 can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human beta-protakinin 9 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit human beta-protakinin 9 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that specifically breaks down specific RNAs. Its mechanism of action is that the ribozyme molecule specifically hybridizes to a complementary target RNA for endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as the technology for the synthesis of oligonucleotides by solid-phase phosphoramidite chemical synthesis has been widely used.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA sequence has been integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of a nucleic acid molecule, 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.
  • a polynucleotide encoding human beta-protakinin 9 can be used for the diagnosis of diseases related to human beta-protakinin 9.
  • a polynucleotide encoding human beta-protakinin 9 can be used to detect the expression of human beta-protakinin 9 or the abnormal expression of human beta-protakinin 9 in a disease state.
  • the DNA sequence encoding human beta-protakinin 9 can be used to hybridize biopsy specimens to determine the expression of human beta-protakinin 9.
  • Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. These technical methods are all mature technologies that are publicly available, and related kits are commercially available.
  • polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also referred to as a "gene chip") for differential expression analysis and gene diagnosis of genes in tissues.
  • RNA-polymerase chain reaction (RT-PCR) in vitro amplification of human beta-protakinin 9 specific primers can also detect human beta-pretakinin 9 transcripts.
  • Detection of mutations in the human beta-protakinin 9 gene can also be used to diagnose human beta-pretakinin 9-related diseases.
  • the forms of human beta-protakinin 9 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type human beta-protakinin 9 DNA sequences. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence tillering, 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 marking chromosome positions.
  • an important first step is to locate these DNA sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • sublocalization can be achieved by a similar method using a set of fragments from a specific chromosome or a large number of genomic clones.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the 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 cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • 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 regulatory agencies that manufacture, use or sell pharmaceuticals or biological products, which mentions that the government regulatory agency that manufactures, uses, or sells permits their administration on the human body .
  • 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 beta-protakinin 9 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dose range of human beta-protakinin 9 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 β-préprotachykinine humaine 9, 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 carcinomes du larynx, des troubles du système nerveux, de l'endocrinopathie, des maladies immunitaires, des inflammations, de toutes sortes de tumeurs malignes, des troubles du développment et de la croissance, de l'hémopathie et de l'infection par VIH. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la β-préprotachykinine humaine 9.
PCT/CN2001/000188 2000-03-02 2001-02-26 Nouveau polypeptide, $g(b)-preprotachykinine humaine 9, et polynucleotide codant pour ce polypeptide Ceased WO2001074888A1 (fr)

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FEBS LETT., vol. 208, no. 1, 1986, pages 67 - 72 *
J. NEUROSCI., vol. 10, no. 7, 1990, pages 2203 - 2214 *
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