WO2001081389A1 - Nouveau polypeptide, proteine humaine 9 contenant un fragment de sequence particulier d'une proteine ribosomale l19, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, proteine humaine 9 contenant un fragment de sequence particulier d'une proteine ribosomale l19, et polynucleotide codant pour ce polypeptide Download PDF

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WO2001081389A1
WO2001081389A1 PCT/CN2001/000578 CN0100578W WO0181389A1 WO 2001081389 A1 WO2001081389 A1 WO 2001081389A1 CN 0100578 W CN0100578 W CN 0100578W WO 0181389 A1 WO0181389 A1 WO 0181389A1
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protein
polypeptide
polynucleotide
human
sequence
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PCT/CN2001/000578
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English (en)
Chinese (zh)
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Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
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Priority to AU68898/01A priority Critical patent/AU6889801A/en
Publication of WO2001081389A1 publication Critical patent/WO2001081389A1/fr

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

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, a human protein 9 containing a characteristic sequence fragment of ribosomal protein L19, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique
  • Ribosome is one of the main components of the cell. It continuously moves along the mRM in the organism to synthesize peptide chains at an extremely fast speed. It is the main place for the organism to translate mRM into protein polypeptides. Ribosomal proteins are combined with rRNA and proteins in a certain order, and assembled into two ribosomal subunits, a large subunit and a small subunit, where the large subunit is approximately twice the small subunit, and r.RNA directly Involved in the binding of mRNA and tRNA. The ribosome and its cofactors combine to possess all the enzyme activities required at each stage of protein synthesis. These enzyme activities are only available in the presence of the overall ribosome structure, and neither ribosome proteins nor rRNA can participate in protein synthesis reactions alone.
  • ribosome is composed of two large and small subunits.
  • Various ribosome proteins have certain positions in the large and small subunits, and rRNA is the backbone of them. Some ribosomal proteins bind directly to rRNA. After binding these proteins to rRNA, the conformation of rRNA is changed, so that other proteins bind to rRNA again, which is an important component of maintaining ribosome conformation; while some ribosomal proteins do not directly interact with rRNA.
  • rRNA binds, but binds to other proteins. These proteins work in concert with other proteins in the body to enable the ribosome to perform normal physiological functions. All ribosomal proteins constitute an independent protein family, the ribosomal protein family.
  • Ribosomal protein L19 is also an important ribosomal protein in the body, which is an important component of the large ribosomal subunit. All L19 proteins consist of approximately 148 to 203 amino acid residues. The protein binds to small rRNAs in the ribosomal 60S subunit in the body to form a catalytic extension complex. It synergizes with the transcription and translation of proteins in the ribosome in vivo to regulate the transcription and translation of related proteins in vivo [Yuen-Ling Chan, Alan L in et al., 1987, J Bio l. Chem., 262: 1111-1115]. The mutation or abnormal expression of this protein may be related to the occurrence of some metabolic disorders and some tumors and cancers.
  • Sequence fragment Q— [KR] — R— [LIVM] — X— [SA] — X (4)-[CV] -GX (3)-[IV]-[K]-[LIVF]-[DNS] -P; This sequence fragment is an important active site for mRNA transcription and translation elongation factors that binds small rRM and 5S and 5.8S rRNAs in the ribosomal 60S subunit.
  • ribosomal protein L19 is an important protein in the process of protein synthesis and extension in vivo. Its mutation or abnormal expression is related to disorders of metabolic regulation, immune system diseases and various tumors and cancers. The occurrence is closely related.
  • the protein 9 protein containing the human ribosomal protein L19 characteristic sequence fragment plays an important role in important functions in the body, and it is believed that a large number of proteins are involved in these regulatory processes, so it is always necessary to identify more involved in these Process of human protein 9 protein containing a characteristic sequence fragment of ribosomal protein L19, especially the amino acid sequence of this protein is identified.
  • Isolation of the protein 9 gene encoding the ribosomal protein L19 characteristic sequence of the newcomer also provided a basis for the study to determine the role of the protein in health and disease states. This protein may form the basis for developing diagnostic and / or therapeutic drugs for the disease, so isolating its coding DNA is important. 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 protein 9 encoding a human ribosome protein-containing L19 characteristic sequence fragment.
  • Another object of the present invention is to provide a genetically engineered host cell comprising a polynucleotide encoding a protein 9 encoding a human ribosome protein-containing L19 characteristic sequence fragment.
  • Another object of the present invention is to provide a method for producing a protein 9 containing a human ribosomal protein L19 characteristic sequence fragment.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention-human protein 9 containing a characteristic sequence fragment of ribosomal protein L19.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to protein 9 of the polypeptide of the present invention, including human ribosomal protein L19 characteristic sequence fragments.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormality of protein 9 containing human ribosome protein L19 characteristic sequence fragment.
  • the present invention relates to an isolated polypeptide.
  • the polypeptide is of human origin and comprises: SEQ ID No. 2 Amino acid sequence of a polypeptide, 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 2159-241 in SEQ ID NO: 1; and (b) having a sequence in SEQ ID NO: 1 in 1- 2643-bit sequence.
  • the invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • a vector in particular an expression vector, containing the polynucleotide of the invention
  • a host cell genetically engineered with the vector including a transformed, transduced or transfected host cell
  • a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the protein 9 protein activity of human ribosomal protein L19 characteristic sequence fragments, which comprises utilizing the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for detecting a disease or disease susceptibility related to abnormal expression of protein 9 protein containing human ribosome protein L19 characteristic sequence fragment in vitro, which comprises detecting the polypeptide or a polynucleotide sequence encoding the same in a biological sample. Mutations, or the amount or biological activity of a polypeptide of the invention in a biological sample.
  • 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 polypeptides and / or polynucleotides of the present invention for the treatment of cancer, developmental disease or immune disease or other diseases caused by abnormal expression of protein 9 containing human ribosomal protein L19 characteristic sequence fragments. Use of medicine.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to the complete natural ammonia related to the protein molecule Based acid.
  • a protein or polynucleotide “variant” refers to an amino acid sequence having one or more amino acid or nucleotide changes or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. Variants can have "conservative" changes in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as 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 can cause changes in the protein and thereby regulate the activity of the protein when combined with protein 9 containing human ribosomal protein L19 characteristic sequence fragments.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that binds to a human protein 9 containing a characteristic sequence of ribosomal protein L19.
  • Antagonist refers to an organism that can block or regulate protein 9 containing a characteristic sequence fragment of human ribosomal protein L19 when combined with protein 9 containing a characteristic sequence fragment of human ribosomal protein L19.
  • Molecularly active or immunologically active molecule may include proteins, nucleic acids, carbohydrates, or any other molecule that binds to protein 9 containing human ribosomal protein L19 characteristic sequence fragments.
  • Regular refers to a change in the function of human protein 9 containing a characteristic sequence fragment of ribosomal protein L19, including an increase or decrease in protein activity, a change in binding characteristics, and human protein 9 containing a characteristic sequence fragment of ribosomal protein L19. Of any other biological, functional or immune properties.
  • substantially pure ' means essentially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated.
  • Those skilled in the art can use standard protein purification techniques to purify human ribosome-containing protein L19 characteristic sequences Fragment of protein 9.
  • Essentially pure human ribosome protein L19 characteristic sequence fragment of protein 9 can generate a single main band on a non-reducing polyacrylamide gel.
  • Human ribosome protein L19 characteristic sequence fragment The purity of the protein 9 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to polynucleotides that naturally bind through base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence C-GA
  • complementary sequence G-A-C-T
  • Two orders The complementarity between the chain molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits the hybridization of a fully complementary sequence to a target nucleic acid. The inhibition of such hybridization can be detected by performing hybridization (Southern or Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that two sequences bind to each other as a specific or selective interaction.
  • 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 through the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Clus ter method (Hi gg ins, D. G. and P. M. Sharp (1988) Gene 73: 237-244). The Cluster method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: The number of matching residues between sequence A and sequence X 100
  • nucleic acid sequences can also be determined by the Cluster method or by a method known in the art such as Jotun He in Percentage (He in J., (1990) Methods in emzumo logy 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 encode codes that retain the main biological properties of natural molecules Peptide.
  • 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 protein 9 of human ribosome protein L19 characteristic sequence fragments.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it occurs naturally).
  • a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist 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 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 ribosome protein L1 9 characteristic sequence fragment-containing protein 9 means that human ribosome protein L19 characteristic sequence fragment-containing protein 9 is substantially free of other proteins and lipids naturally associated with it. , Sugar or other substances. Those skilled in the art can use standard protein purification techniques to purify protein 9 containing human ribosomal protein L19 characteristic sequence fragments. Substantially pure polypeptides produce a single main band on a non-reducing polyacrylamide gel. The purity of human protein 9 peptide containing a characteristic sequence fragment of ribosomal protein L19 can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human protein 9 containing a characteristic sequence fragment of ribosomal protein L19, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques.
  • polypeptides of the invention may be glycosylated, or they may be non-glycosylated.
  • the polypeptides of the invention may also include or exclude the initial methionine residue.
  • the present invention also includes fragments, derivatives, and analogs of human protein 9 containing a characteristic sequence fragment of ribosomal protein L19.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of protein 9 of the human ribosome-containing protein L19 characteristic sequence fragment of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: U) such a kind, in which one or more The amino acid residue is replaced by a conservative or non-conservative amino acid residue (preferably a conservative amino acid residue), and the substituted amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) such one or more A group on an amino acid residue is substituted by another group to include a substituent; or (in) a type in which a mature polypeptide is fused to another compound (such as a compound that extends the half-life of a polypeptide, such as polyethylene glycol) Or (IV) a polypeptide sequence (such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protease sequence) formed by fusing additional amino acid sequences into a mature polypeptide, as described herein, such that Fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes a nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a CDM library of human fetal brain tissue. It contains a polynucleotide sequence of 2643 bases in length and its open reading frame (2159-2410) encodes 83 amino acids.
  • This polypeptide has the characteristic sequence of the ribosomal protein family, and it can be deduced that the human protein 9 containing the characteristic sequence fragment of ribosomal protein L19 has the structure and function represented by the ribosomal protein family.
  • 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 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide that encodes the polypeptide and a polynucleotide that includes additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide may 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) at lower ionic strength and higher Hybridization and elution at temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) adding a denaturing agent, such as 50 ° / »(v / v) formamide, 0.1 % Calf serum / 0.1 ° /.
  • 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 protein 9 of human ribosome-containing protein L19 characteristic sequence fragments.
  • 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 of the protein 9 encoding the human ribosome-containing protein L19 characteristic sequence fragment 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 DM 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 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 (Q i agene).
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Moleculo r Cloning, A Labora tory Manual, Collspring 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 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-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of protein 9 of human ribosome protein L19 characteristic sequence fragments The level of transcripts; (4) Detecting protein products expressed by genes by immunological techniques or by measuring biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides. It is preferably within 1000 nucleotides.
  • the probe used herein is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention. The genes or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product that detects the expression of the protein 9 gene of human ribosome L19 characteristic sequence fragments can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). Wait.
  • a method of applying a PCR technique to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE- rapid cDNA end amplification method
  • 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 DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, the sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a protein 9 coding sequence containing human ribosomal protein L19 characteristic sequence fragments, and produced by recombinant technology.
  • a method of a polypeptide according to the invention is 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 protein 9 coding sequence containing human ribosomal protein L19 characteristic sequence fragments, and produced by recombinant technology.
  • a polynucleotide sequence encoding a protein 9 containing a human ribosomal protein L19 characteristic sequence fragment can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, bacteriophages, 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 replication origins, promoters, marker genes, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing the DNA sequence of protein 9 encoding human ribosome-containing protein L19 characteristic sequence fragments and suitable transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manua l, cold Spr ing Harbor Labora tory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the l ac or trp promoter of E.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenovirus enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, 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 protein 9 containing a characteristic sequence fragment of ribosomal protein L19 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a gene containing the polynucleotide or the recombinant vector.
  • Genetically engineered host cells refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • Escherichia coli, Streptomyces bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells insect cells
  • fly S2 or Sf 9 animal cells
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of DNA uptake can be in the exponential growth phase were harvested, treated with CaC l 2 method used in steps 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 ribosome protein L19-containing characteristic sequence fragment protein 9 (Scence, 1984; 224: 1431). Generally there are the following steps: (1) Use the polynucleotide (or variant) of protein 9 encoding a human-human ribosome-containing protein L19 characteristic sequence fragment of the present invention, or transform or transduce a suitable expression vector using a recombinant expression vector containing the polynucleotide Host cell
  • the medium used in the culture may be selected from various conventional mediums according to the host cells used. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be 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 amino acid sequence homology of 49 amino acids and characteristic domains of the ribosomal protein family of protein 9 of the inventor's ribosome protein L19 characteristic sequence fragment at 13-61.
  • the upper sequence is human protein 9 containing a characteristic sequence fragment of ribosomal protein L19, and the lower sequence is the characteristic domain of the ribosomal protein family.
  • Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+".
  • Figure 1 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of human protein 9 containing a characteristic sequence fragment of ribosomal protein L19.
  • 9KDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • the Quik mRNA Isolation Kit (Qiegene) was used to isolate poly (A) mRNA from total RNA. 2ug poly (A) mRNA was reverse transcribed to form cDNA.
  • a Smart cDNA cloning kit (purchased from Clontech) was used to insert the 00 ⁇ fragment into the multiple cloning site of the P BSK (+) vector (Clontech) to transform DH5 ⁇ to form a cDNA library.
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public DM sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0944e05 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • sequence of protein 9 of the human ribosome-containing protein L19 characteristic sequence fragment and the encoded protein sequence J 1 J of the present invention were used in a profiling scan program (Basiclocal Alignment search in GCG)
  • the human ribosome protein L19-containing characteristic sequence fragment protein 9 of the present invention is
  • 13-61 is homologous to the domain ribosomal protein family. The homology results are shown in Figure 1. The homology is 0.31, the score is 15.03; the threshold is 13.35.
  • Example 3 Cloning of the gene encoding protein 9 of human ribosome-containing protein L19 characteristic sequence fragment 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:
  • Primer 1 5-GTGTAAAATTTGTATATTTGCATT-3 '(SEQ ID NO: 3)
  • Priraer2 5'-AGCAGTTCATTTTATTGTCCATTA-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 mmol / L KC1, 10 mmol / L in a 50 ⁇ l reaction volume Tris-Cl, (pH 8.5), 1.5 mmol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol primer, 1U Taq 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.
  • ⁇ -act in 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 using a TA cloning kit (Invitrogen). DM sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1-2643bp shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of the protein 9 gene expression of human ribosomal protein L19 characteristic sequence fragments:
  • 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 ) 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.
  • Primer3 5-CATGCTAGCATGATCACCAAGTTTAGTGAACTT-3 '(Seq ID No: 5)
  • Priraer4 5'- CCCGAGCTCTTATCCTTTAGGATGTGGTATTTG- 3 '(Seq ID No: 6)
  • the 5' ends of these two primers contain Nhel and Sacl digestion sites, respectively, followed by the coding sequences of the 5, 5, and 3 'ends of the target gene.
  • Nhel and Sacl restriction sites correspond to selective endonuclease sites on the expression vector plasmid pET 28b (+) (Novagen, Cat. No. 69865.3).
  • the PCR reaction was performed using pBS-0944e05 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions are: Total volume 50 ⁇ ⁇ pBS- 0944e05 plasmid 10pg, 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. Nhel and Sacl were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • Ligation products were transformed by the calcium chloride method Escherichia bacteria DH5 C, after (final concentration of 30 ⁇ ⁇ / ⁇ 1) LB plates incubated overnight positive clones by colony PCR method containing kanamycin, and sequenced.
  • a positive clone (pET-0944e05) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) P lySs (product of Novagen) using the calcium chloride method.
  • the host strain BL21 (pET-0944e05) was at 37 in LB liquid medium containing kanamycin (final concentration 30 ⁇ ⁇ / ⁇ 1). C.
  • a peptide 9-specific peptide containing the characteristic sequence fragment of human ribosomal protein L19 was synthesized using a peptide synthesizer (product of PE company):
  • a titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum.
  • Total IgG was isolated from antibody-positive rabbit sera using protein A-Sepharose.
  • the peptide was bound to a cyanogen bromide-activated Sephar 0S e4B column, and the anti-peptide antibody was separated from the total IgG by affinity chromatography.
  • the immunoprecipitation method proved that the purified antibody could specifically bind to protein 9 containing human ribosomal protein L19 characteristic sequence fragment.
  • Example 7 Use of a polynucleotide fragment of the present invention as a hybridization probe
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to Identifying whether it contains the polynucleotide sequence of the present invention and detecting a homologous polynucleotide sequence, further The probe is used to detect whether the expression of the polynucleotide sequence of the present invention or a homologous polynucleotide sequence thereof in cells of normal tissues or pathological tissues 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, Nor thern blotting, and copying methods, etc. They are all used to fix the polynucleotide sample to be tested on the filter and then hybridize using basically the same steps.
  • 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 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 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 fragments from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • the GC content is 30% -70%, and the non-specific hybridization increases when it exceeds;
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, then the primary probe should not be used;
  • Probe 1 ( pr 0 bel), 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 belongs to the second type of probe and is equivalent to the gene fragment of SEQ ID NO: 1 or its mutual Replacement mutation sequence of complement 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 a 3-1 Omg pre-hybridization solution (lOxDenhardfs; 6xSSC, 0.1 mg / ml) was added.
  • CT DNA calf thymus DNA).
  • 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.
  • Ribosomes and other cofactors together provide the full enzymatic activity of the translation process. These enzyme activities are only in the ribosome. Only if the overall structure is complete. The ribosome size subunits are often free in the cytoplasmic matrix in the cell. Only when the small subunits are combined with the mRNA can the large subunits combine with the small subunits to form a complete ribosome and initiate the synthesis of the peptide chain.
  • Ribosomal protein L19 is one of the proteins in the large ribosomal subunit, which plays a very important role in correct tRNA selection in the initial stage of protein synthesis L19 is very important for rRM to fold into a functional three-dimensional structure. It plays an important role in the correct translation of proteins.
  • the characteristic sequence of the ribosomal protein L19 family is necessary for its biological activity.
  • the polypeptide of the present invention is a polypeptide containing a characteristic sequence of this protein family. Abnormal expression of the polypeptide will cause abnormal interaction of various aminoacyl-tRNA synthetases, various tRNAs, and ribosomes, and cause abnormal ribosome translation functions, and produce Related diseases.
  • abnormal expression of protein 9 of the human ribosomal protein L19-containing characteristic sequence fragment of the present invention will produce various diseases, especially various tumors, embryonic development disorders, and growth disorders. These diseases include, but are not Limited to:
  • Fetal developmental disorders congenital abortion, cleft palate, facial oblique fissure, limb absentness, limb differentiation disorder, gastrointestinal atresia or stenosis, hyaline membrane disease, pulmonary insufficiency, polycystic kidney disease, ectopic kidney, double ureter, crypto, Congenital inguinal hernia, double uterus, vaginal atresia, hypospadias, hermaphroditism, atrial septal defect, ventricular septal defect, pulmonary stenosis, arterial duct closure, neural tube defect, congenital hydrocephalus, iris defect, congenital cataract , Congenital glaucoma or cataract, congenital deafness
  • Growth and development disorders mental retardation, cerebral palsy, brain development disorders, mental retardation, familial cerebral nucleus dysplasia syndrome, strabismus, skin, fat and muscular dysplasia such as congenital skin laxity, premature aging Disease, congenital keratosis, various metabolic defects such as various amino acid metabolic defects, stunting, dwarfism, sexual retardation
  • Tumors of various tissues gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioblastoma, Colon cancer, malignant histiocytosis, bladder cancer, bone cancer, osteosarcoma, myeloma, bone marrow cancer, brain cancer, uterine cancer, endometrial cancer, gallbladder cancer, colon cancer, thymic tumor, nasal cavity and sinus tumor, nasopharynx Cancer, laryngeal cancer, tracheal tumor, pleural mesothelioma, fibroid, fibrosarcoma, lipoma, liposarcoma, leiomyoma
  • the abnormal expression of the protein 9 containing the characteristic sequence fragment of the ribosomal protein L19 of the present invention will also cause certain hereditary, hematological and immune system diseases.
  • the polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially embryonic developmental disorders, growth and development disorders, various tumors, inflammation, certain diseases. Some hereditary, hematological and immune system diseases.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) protein 9 of human ribosomal protein L19 characteristic sequence fragments. Agonists enhance biological functions such as human ribosome protein L19 characteristic sequence fragments to stimulate cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • a mammalian cell or a membrane preparation expressing protein 9 containing a characteristic sequence fragment of human ribosomal protein L19 can be cultured with a labeled human ribosome protein L19 characteristic sequence fragment-containing protein 9 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of protein 9 containing human ribosomal protein L19 characteristic sequence fragments include screened antibodies, compounds, receptor deletions, and the like.
  • An antagonist of human protein 9 containing a characteristic sequence fragment of ribosomal protein L19 can bind to human protein 9 containing a characteristic sequence fragment of ribosomal protein L19 and eliminate its function, or inhibit the production of the polypeptide, or with the polypeptide The active site binding prevents the polypeptide from performing biological functions.
  • protein 9 of human ribosome protein L19 characteristic sequence fragment can be added to the bioanalytical assay, and the protein 9 of human ribosome protein L19 characteristic sequence fragment and its receptor can be determined by measuring the compound.
  • 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 9 containing a characteristic sequence fragment of ribosomal protein L19 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. During screening, generally, 9 proteins of human ribosomal protein L19 characteristic sequence fragments 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 directed against the protein 9 epitope of human ribosomal protein L19 characteristic sequence fragments. 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 directly injecting human 9 containing ribosomal protein L19 characteristic sequence fragments 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 it is not limited to Freund's adjuvant.
  • Techniques for preparing human monoclonal antibodies that contain fragments of the ribosomal protein L19 characteristic sequence include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology , Human B-cell hybridoma technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions and non-human-derived variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851).
  • the existing technology for producing single chain antibodies (US Pat. No. 4946778) can also be used to produce single protein 9 against human ribosome protein L19 characteristic sequence fragments. Chain antibodies.
  • Antibodies against protein 9 containing a characteristic sequence fragment of ribosomal protein L19 can be used in immunohistochemistry to detect protein 9 containing a characteristic sequence fragment of human ribosomal protein L19 in a biopsy specimen.
  • Monoclonal antibodies that bind to protein 9 containing human ribosomal protein L19 characteristic sequence fragments can also be labeled with radioisotopes and injected into the body to track their location and distribution.
  • This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • High affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill the characteristic sequence of human ribosomal protein L19 Fragment of protein 9 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to protein 9 containing human ribosomal protein L19 characteristic sequence fragments.
  • Administration of an appropriate dose of the antibody can stimulate or block the production or activity of protein 9 containing human ribosomal protein L19 characteristic sequence fragments.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the protein 9 level of a human ribosome protein L19 characteristic sequence fragment.
  • tests are well known in the art and include FI SH assays and radioimmunoassays.
  • the level of protein 9 of human ribosomal protein L19 characteristic sequence fragments detected in the test can be used to explain the importance of human ribosome protein L19 characteristic sequence fragments of protein 9 in various diseases and to diagnose humans Diseases in which protein 9 containing a characteristic sequence fragment of ribosomal protein L19 functions.
  • 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.
  • Polynucleotides encoding protein 9 of human ribosomal protein L19 characteristic sequence fragments 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 protein 9 containing human ribosomal protein L19 characteristic sequence fragments.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human ribosome protein L19 characteristic sequence fragment protein 9 to inhibit endogenous human ribosome protein L19 characteristic sequence fragment protein 9 active.
  • a mutated human ribosome protein L19 characteristic sequence fragment protein 9 may be a shortened human ribosome protein L19 characteristic sequence fragment protein 9 lacking a signaling domain, although it can be related to downstream Substrate binding, but lacks signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of protein 9 containing human ribosomal protein L19 characteristic sequence fragments.
  • Virus-derived expression vectors such as retroviruses, adenoviruses, adenovirus-associated viruses, herpes simplex virus, parvoviruses, and the like can be used to encode a polynucleoside of protein 9 encoding human ribosome protein L19 characteristic sequence fragments
  • the acid is transferred into the cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a protein 9 containing a human ribosomal protein L19 characteristic sequence fragment can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human protein 9 containing a characteristic sequence fragment of ribosomal protein L 19 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 protein 9 mRNA containing fragments characteristic of ribosomal protein L19 are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technology. For example, solid-phase phosphate amide chemical synthesis to synthesize oligonucleotides 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 the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the phosphorothioate or peptide bond is used instead of the phosphodiester bond to link the ribonucleosides.
  • the polynucleotide encoding the protein 9 containing the human ribosome protein L19 characteristic sequence fragment can be used for the diagnosis of diseases related to the human ribosome protein L19 characteristic sequence fragment-containing protein 9.
  • the polynucleotide encoding protein 9 of human ribosome protein L19 characteristic sequence fragment can be used to detect the expression of human ribosome protein L19 characteristic sequence fragment protein 9 or human ribosome protein L 1 in disease state Abnormal expression of protein 9 with 9 characteristic sequence fragments.
  • a DNA sequence encoding protein 9 containing human ribosome protein L 19 characteristic sequence fragments can be used to hybridize biopsy specimens to determine the expression of protein 9 containing human ribosome protein L 1 9 characteristic sequence fragments.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are all mature and open technologies, and related kits are commercially available. Part or all of the polynucleotides of the present invention can be used as probes to be fixed on a micro array (Mic roar ray) or a DM chip (also known as a "gene chip"), and used to analyze differential expression analysis of genes and genes in tissues. diagnosis.
  • RNA-polymerase chain reaction (RT-PCR) in vitro amplification of human ribosome protein L19 characteristic sequence fragment protein 9 specific primers can also detect human ribosome protein L1 9 characteristic sequence fragment protein 9 transcription product.
  • Detecting mutations in the protein 9 gene of human ribosome protein L19 characteristic sequence fragments can also be used to diagnose human protein 9-containing disease sequence containing ribosomal protein L19 characteristic sequence fragments.
  • Human ribosome-containing protein L 1 9 characteristic sequence fragment of the protein 9 mutant form includes characteristics similar to normal wild-type human ribosome-containing protein L1 9 Sex sequence fragments of the protein 9 DNA sequence compared to point mutations, translocations, deletions, recombinations and any other abnormalities. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • 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.
  • the PCR primers (preferably 15-35bp) are prepared according to cDM, and the sequences can be located on the 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 DM to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct chromosome-specific cDM libraries.
  • Fluorescent in situ hybridization of cDM clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mende l an an inher tance in Man (available online with Johns Hopk ins University Welch Med ica l Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the cD or genomic sequence differences between the affected and unaffected individuals need to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, 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 protein 9 containing a characteristic sequence fragment of ribosomal protein L19 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dose range of human ribosome L19 characteristic sequence fragment-containing protein 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 protéine humaine 9 contenant un fragment de séquence particulier d'une protéine ribosomale L19, et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, des troubles du développement, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la protéine humaine 9 contenant un fragment de séquence particulier d'une protéine ribosomale L19.
PCT/CN2001/000578 2000-04-27 2001-04-23 Nouveau polypeptide, proteine humaine 9 contenant un fragment de sequence particulier d'une proteine ribosomale l19, et polynucleotide codant pour ce polypeptide WO2001081389A1 (fr)

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CN00115493.1 2000-04-27
CN 00115493 CN1320639A (zh) 2000-04-27 2000-04-27 一种新的多肽——人含核糖体蛋白l19特征性序列片段的蛋白9和编码这种多肽的多核苷酸

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DAVIES B. ET AL.: "The L19 ribosomal protein gene (RPL19): gene organization, chromosomal mapping and novel promoter region", GENOMICS, vol. 25, no. 2, 20 January 1995 (1995-01-20), pages 372 - 380 *
HENRY J.L. ET AL.: "High-level expression of the ribosomal protein L19 in human breast tumors that overexpress erbB-2", CANCER RES., vol. 53, no. 6, 15 March 1993 (1993-03-15), pages 1403 - 1408 *

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