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WO2001073013A1 - Nouveau polypeptide, proteine ribosomale s7 humaine 16, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, proteine ribosomale s7 humaine 16, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001073013A1
WO2001073013A1 PCT/CN2001/000528 CN0100528W WO0173013A1 WO 2001073013 A1 WO2001073013 A1 WO 2001073013A1 CN 0100528 W CN0100528 W CN 0100528W WO 0173013 A1 WO0173013 A1 WO 0173013A1
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protein
polypeptide
polynucleotide
human
sequence
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PCT/CN2001/000528
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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 AU73781/01A priority Critical patent/AU7378101A/en
Publication of WO2001073013A1 publication Critical patent/WO2001073013A1/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 ribosomal S7 protein 16, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique
  • the correct translation of proteins is very important for all bacteria and higher organisms.
  • the research on the regulation mechanism of protein translation is obtained from E. coli.
  • the protein translation process is mainly completed by various aminoacyl-tRNA synthetases, various tRNAs, and ribosomes.
  • the ribosome and other cofactors together provide the full enzymatic activity of the translation process. Ribosomes are only available if the entire crust is intact. Therefore, the ribosome and its constituent subunits act synergistically in the body and play an important physiological function.
  • Ribosomes are organelles that synthesize proteins. Their sole function is to synthesize polypeptide chains from amino acids in accordance with the instructions of mRNA. It is called ribosome, and is simply called ribosome or ribosome. Ribosomes are found in almost all cells, whether in prokaryotic or eukaryotic cells, there are a large number of ribosomes. The ribosome is a granular structure without a membrane. Its diameter is 25 nm. The main components are protein and RNA. Ribosomal RNA is called rRNA. The protein content is about 40% and RM is about 60%.
  • Protein molecules are mainly distributed on the surface of the ribosome, while rRNA is located inside, and the two are bound together by non-covalent bonds.
  • Cell Biology Szhonghe Higher Education Press PP122-129 The function of ribosomes has focused on the ribosome R'A. There are many rRNA functional domains that determine different functions of the ribosome. (Annu Rev Biochem 1991; 60 191-227)
  • ribosomes There are two basic types of ribosomes in biological organism cells: one is a 70S (S is the Sverdberg sedimentation coefficient unit) ribosome, and all prokaryotic cells are 70S ribosomes in eukaryotic cells. Approximately 70S. The other is 80S ribosomes. The ribosomes of eukaryotic cells (except for mitochondria and chloroplast ribose) are 80S. Ribosomes, whether 70S or 80S, are composed of two subunits of different sizes.
  • the ribosome size subunits are often free in the cytoplasmic matrix within the cell. Only when the small subunits are combined with mRNA do the large subunits bind to the small subunits to form a complete ribosome. After the peptide synthesis is terminated, the large and small subunits dissociate and then exist freely in the cytoplasmic matrix.
  • S7 is one of the small subunits of ribosomal binding protein, and it exists in most prokaryotic cells with homologous proteins Biological and eukaryotic cells.
  • All ribosomal eggs contain a conserved region at the N-terminus of S7, which contains the following consensus sequence fragments: [DENSK]-X- [LIVMET] — X (3) [LIVMFT] (2) - ⁇ (6 ) -G--[KR] - ⁇ (5)-[LIVMF]-[LIVMFC] -x (2)-[STA].
  • This sequence fragment is contained in ribosomal protein S7 in many different organisms.
  • This conserved structural motif is of great significance for the binding process of ribosomal protein S7 and ribosomal RNA, and this fragment may play a normal role in ribosomes. Physiological functions play an important regulatory role.
  • ribosomal protein S7 can directly bind to the 3 'end of 16S ribosomal RNA.
  • Ribosomal protein S7 plays an important role in the translation process of proteins. If it is deleted or chemically modified, or the gene encoding it is mutated, it will affect the function of ribosomes and reduce the activity of peptide synthesis. Although the research on the function of ribosomal protein S7 is not thorough enough, according to various research results, it can be proved that the functions of ribosomal protein S7 include: (1) It is very important for rRNA to fold into a functional three-dimensional structure; (2 ) In protein synthesis, the spatial conformation of the ribosome undergoes a series of changes, and ribosomal white may play a "fine-tuning" role in the conformation of the ribosome; (3) it may even play a catalytic role in the ribosome binding site, Ribosome proteins work together with rRNA.
  • ribosomal protein S7 Based on the sequence similarity of ribosomal protein S7, it is considered to belong to the ribosomal protein family. Deletion of this protein will slow down cell growth [] ⁇ Stephen Gantt, Michael D. Thompson, 1990, J. Biol. Chem., 265: 2763-2767] 0 Therefore, ribosomal protein S7 is transcribed and translated from DNA It plays an extremely important role in cell proliferation. Its abnormal expression will cause the growth rate of the tissue to slow down, which will cause various developmental disorders, such as stunting.
  • the expression profile of the present invention is very similar to the expression profile of human ribosomal S7 protein 9, and the two functions may be similar.
  • the invention is named human ribosomal S7 protein 16.
  • the human nuclear S7 protein 16 protein plays an important role in regulating important functions of the body such as cell division and fetal development, and it is believed that a large number of proteins are involved in these regulatory processes, so more needs to be identified in the field.
  • the human ribosomal S7 protein 16 protein involved in these processes especially the identification of the amino acid sequence of this protein.
  • the isolation of the new human ribosome S7 protein 16 protein encoding gene also provides a basis for studying the role of this protein in health and disease states. . This protein may form the basis for the development of diagnostic and / or therapeutic agents for the disease. 21 It is important to isolate its coding DNA. Disclosure of invention
  • the present invention also provides a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a human nucleosome S7 protein 16.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human ribosome S7 protein 16.
  • Another object of the present invention is to provide a method for producing human ribosomal S7 protein 16.
  • Another object of the present invention is to provide an antibody against the polypeptide-human ribosomal S7 protein 16 of the present invention.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors of the polytitanium-human ribosomal S7 protein 16 of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of human ribosomal S7 protein 16.
  • the invention includes a step and an isolated polypeptide, the polypeptide is human: it includes: a polypeptide having the amino acid sequence of SEQ ID No. 2 or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence of SEQ ID NO: 1 with a sequence of 80 and 122 ⁇ M ⁇ ?; and (b) a sequence with SEQ ID NO: i- A sequence of 1-2533 bits;
  • the present invention further relates to a vector, particularly an expression vector, containing the polynucleoside 3 ⁇ 4 of the present invention; a host cell engineered with the vector, including a transformed, transduced or transfected host cell; The method for preparing the main cell and recovering the expressed product is described.
  • the present invention relates to an antibody capable of specifically binding to the polypeptide of the present invention.
  • the present invention relates to a screening method for mimicking, activating, anti-or inhibiting or inhibiting the activity of the human ribosome S7 protein 16 protein, which includes the use of the present invention.
  • the invention also relates to compounds obtained by this method.
  • the present invention also relates to a method for in vitro detection of diseases or susceptibility to diseases associated with abnormal expression of human ribosomal S 7 protein 16 protein, comprising detecting mutations in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample. Or Detecting the amount or biological activity of the polypeptide of the present invention in a biological product.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the use of the polyamidine and / or polynucleotide of the present invention in the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of human ribosomal S 7 protein 16.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide, or a polynucleotide and a fragment or part thereof, and may also refer to the genome Or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a protein or polynucleotide “variant” refers to an amino acid sequence or a polynucleotide sequence that has one or more amino acids or nucleotide changes.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide string.
  • the X-body may have "conservative" changes, in which the substituted amino acid has similar structural or chemical properties as the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” means the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence
  • Insertion means that a change in the amino acid sequence or nucleotide sequence 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 A amino acids or nucleotides by different amino acids or nucleotides.
  • Bioactivity refers to proteins that have the structure, regulation, or biochemical function of natural molecules.
  • immunological activity refers to the ability of natural, recombinant, or synthetic white matter and fragments thereof to induce a specific immune response in appropriate animals or cells and to bind to specific antibodies.
  • Agitated 'r' means when bound to '16 Glycoprotein S 7 protein. One can cause changes in this protein A molecule that regulates the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to human ribosomal S7 protein 16.
  • Antagonist refers to a molecule that, when combined with human ribosome S7 protein 16, can block or regulate the biological or immunological activity of human ribosome S7 protein 16.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that binds human ribosomal S7 protein 16.
  • Regular refers to a change in the function of human ribosome S7 protein 16, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological, functional, or immune properties of human ribosome S7 protein 16.
  • 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 ribosomal S7 protein 16 using standard protein purification techniques. Basically Pure human ribosomal S7 protein 16 can generate a single main band on a non-reducing polyacrylamide gel. The purity of human ribosomal S7 protein 16 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence C-T-G-A
  • complementary sequence G-A-C-T.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern imprinting or Northern imprinting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that conditions with reduced stringency allow non-specific binding, because conditions with reduced stringency require the binding of two sequences 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 percentage of identity can be determined electronically, such as through the MEGALIGN program (Lasergene software package, DNASTAR, Inc., Mad Son). Wis.) ; MEGALIGN program can compare two or more sequences according to different methods such as Cluster method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244) 0 Cluster method checks the distance between all pairs The groups of sequences are arranged into clusters. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: Number of residues A matching between sequence A and sequence B
  • the percent identity between nucleic acid sequences can also be determined by the Cluster method or by well-known methods such as Jotun Hein (Hein J., (1990) Methods in emzumology 183: 625-645).
  • Similarity refers to the degree of amino acid sequence: the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment.
  • Amino acids used for conservative substitutions for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino carboxyls may include lysine and arginine; have uncharged head groups
  • Amino acids with similar hydrophilicity 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 ⁇ 'j.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative means HFP or a chemical modification that encodes it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa, F (ab ') 2 and Fv, which can specifically bind to the antigenic determination of human ribosomal S 7 protein 16.
  • Humanized antibody means non-antigen binding :! The amino acid sequence of the domain has been replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of matter from its environment (for example, its natural environment if it occurs naturally).
  • naturally occurring polynucleotides or polypeptides are not isolated in the presence of two living animals, but Tongxiang polyglycosides or peptides are separated from some or all of the substances that coexist in the natural system. of.
  • the polynucleotide of the zenith 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: such as polynucleotides and polypeptides in their natural state in living cells are It is not isolated and purified, but the same polynucleotide or polypeptide is separated from other substances existing in its natural state. It is isolated and pure.
  • isolated human ribose ⁇ S7 protein 16 means that human ribosomal S7 white 16 basically contains other proteins, class 3 ⁇ 4, class 3 ⁇ 4, or other substances naturally associated with it. Skilled person Humans can purify human ribosomal S7 protein 16 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the human ribosomal S7 protein 16 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide-human ribosomal S7 protein 16, 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 invention may be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of the human ribosome S7 protein 16.
  • the terms ",” “derivatives” and “analogs” refer to polypeptides that substantially retain the same biological function or activity of the human ribosome S7 protein 16 of the invention.
  • the analog or analog may be: (I) a type in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substituted amino acid may or may not be Encoded by the genetic codon; or (II) such a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or (in) this type, wherein the mature polypeptide Fusion with another compound (such as a compound that prolongs the half-life of a polypeptide, such as polyethylene glycol); or (IV) a polypeptide sequence in which an additional amino acid sequence is fused into a mature polypeptide, J 'J (as in the leading column Or secreted sequences or sequences used to purify this polypeptide or protease sequences) As explained herein, these fragments, derivatives and similar It is considered within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide). It consists essentially of a polynucleotide encoding a polynucleotide 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 2533 bases, and its open reading frame 801-1229 encodes 142 amino acids.
  • this polypeptide has a similar expression profile with human ribosomal S7 protein 9, and it can be deduced that the human ribosomal S7 protein 16 has similar functions to human ribosomal S7 protein 9.
  • 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.
  • the DNA may be a coding or non-coding strand.
  • the coding region sequence of a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or it may be a variant.
  • “Degenerate variant" in the present invention refers to a protein or polypeptide prepared by SEQ ID NO: 2, ":" blue and the coding sequence shown in SEQ ID NO: 1 Differentiated nucleic acid sequences are listed.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the mature coding sequence and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences) And non-coding sequences.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the present 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 present 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 a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the polypeptides it encodes.
  • the present invention also relates to a polynucleoside carboxylate that hybridizes to the sequence described above (having at least 50. preferably 70% identity between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ion strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) during hybridization Add denaturants, 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 % Or more, more preferably 97. /. Only when the above hybridization occurs.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological energy and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • nucleic acid fragments can also be amplified by nucleic acid amplification technology (such as PCR) to determine and / or isolate the polynucleotide encoding human ribosomal S7 protein 16.
  • 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 ribosomal S7 protein 16 can be obtained by various methods.
  • Example "5. Isolate polynucleotides using hybridization techniques well known in the art. These techniques include but are not limited to: 1) hybridizing a probe to a genomic or cDNA library to detect homologous polynucleotide sequences, and 2) The antibodies of the expression library are screened to detect cloned polynucleotide fragments having common structural characteristics.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) separating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide :
  • genomic DNA is the least commonly used: direct chemical synthesis of DNA sequences Is the method of choice.
  • the more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating cDNA of interest is to isolate mRM from donor cells that overexpress this group 3 and perform reverse transcription to form a plasmid or phage cDNA library.
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Labora tory Manua 1, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction is used in combination, 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 a marker gene function; (3) determination of the transcript of human ribosomal S7 protein 16; (4) ) Detecting protein products of gene expression by immunological techniques or measuring biological activity-The above methods can be used alone or in combination.
  • the probe for heterozygous hearing is homologous to any part of the polynucleotide of the present invention, and has a length of at least 10 nucleosides, preferably at least 30 nucleotides, more Fortunately, at least 50 nucleotides, preferably at least 100 nucleotides. In addition, the length of the probe is usually within 2,000 nucleotides, preferably 1,000 nucleotides.
  • the probe used here is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention. The gene itself or a fragment thereof can of course be used as a probe. DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the detection of: '' the protein product of human ribosomal S7 protein 16 gene expression can be used immunological techniques such as Western blotting, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), etc. .
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers used for PCR can be disclosed according to the present disclosure ⁇ :
  • the polynucleotide sequence of the present invention The information is appropriately selected and synthesized using conventional methods. Isolation and purification of amplified DNA / RNA fragments can be performed by conventional methods: '5 ⁇ by gel electrophoresis.
  • Polynucleotide sequences of the gene of the present invention obtained as described above, or various DNA fragments, etc. can be determined by conventional methods such as dideoxy chain termination: 3 ⁇ 4 (Sanger et al. PNAS, 1977, 74: 5463-5467). Such assays also commercially polynucleotide sequences:: the Sequencing Kit and the like. In order to obtain the full-length cDNA sequence, sequencing must be repeated. Sometimes the cDNA sequence of multiple A clones is needed 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 the human ribosomal S7 protein 16 silk sequence, and the recombinant technology to produce the polypeptide described in the present invention.
  • Party: ' ⁇ In the present invention, a polynucleotide sequence encoding a human ribosomal S7 protein lb may be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al. Gene, 1987, 56: 125) expressed in bacteria; pMSXND expression vectors expressed in mammalian cells ( Lee and Nathans, J Bio Chem. 263: 3521, 1988) and baculovirus-derived vectors expressed in insect cells.
  • 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.
  • 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 DN'A expression, usually about 10 to 300 base pairs, and act on promoters to enhance gene transcription. Examples of Gong include the SV40 enhancer of 100 to 270 base pairs at the late stage of the origin of replication, the polyoma enhancer and the adenovirus enhancer at the late side of the origin of replication.
  • the expression vector preferably contains one A or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and Green fluorescent protein (GFP), or tetracycline or ampicillin against Enterobacteriaceae.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and Green fluorescent protein (GFP), or tetracycline or ampicillin against Enterobacteriaceae.
  • a polynucleotide encoding a human ribosome S7 protein 16 or a recombinant vector containing the polynucleoside can transform or transduce a host cell into a base-engineered host cell containing the polynucleotide or the recombinant vector.
  • the term "primary 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.
  • Streptomyces bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as fly flies S2 or Sf9; animal cells such as CH0, COS or Bowes melanoma cells.
  • 3 ⁇ 4 Transformation of a host cell by the DNA sequence of the present invention or a recombinant vector containing the DNA sequence can be performed by 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 the Ca 2 method. The steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant 3 ⁇ 4 human ribosome S7 protein 16 (Science, 1984; 224: 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. 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 into a cell: if necessary, its physical and chemical properties may be used to isolate and purify the recombinant by various separation methods Of protein.
  • these methods include but are not limited to: conventional renaturation treatment, protein precipitant treatment (3 ⁇ 4 analysis method), centrifugation, osmotic bacteria treatment, 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 (3 ⁇ 4 analysis method)
  • centrifugation osmotic bacteria treatment
  • ultrasonic treatment ultracentrifugation
  • molecular sieve chromatography gel filtration
  • adsorption chromatography Ion exchange chromatography
  • HPLC high performance liquid chromatography
  • 21 is a comparison diagram of gene chip expression profiles of the present human ribosomal S 7 protein 16 and human ribosomal S 7 protein 9.
  • the second figure is the expression profile of the human ribosome S7 protein 16.
  • the figure is the expression profile of the human ribosome S7 protein 9.
  • 1 indicates fetal kidney
  • 2 indicates fetal size ⁇ .
  • 3 indicates ⁇ small intestine
  • 4 indicates fetal muscle
  • 5 indicates brain.
  • 63 ⁇ 4 indicates fetal bladder
  • 7 indicates non-starved L02
  • As 9 indicates ECV304 PMA-.
  • 10 means ECV304 PMA +
  • 11 means fetal liver
  • 12 means normal liver
  • 13 means thyroid
  • 14 means skin
  • 16 means lung
  • 17 means lung cancer
  • 18 means fetal spleen
  • 19 means spleen
  • 20 means prostate
  • 21 is the fetal heart
  • 22 is the heart
  • 23 is the muscle
  • 24 is the testis
  • 25 is the fetal thymus
  • 26 is the thymus.
  • S 2 is a polyacrylamide gel electrophoresis image (SDS-PAGE) of isolated human nuclear sperm S7 protein 16.
  • 16kDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • the determined c DNA sequence was compared with the public DNA word 'f' and 'Database' (Genebank). As a result, one of the clones 0138 a 0 ⁇ c DN A word 'was found: a new sequence of 1
  • the cloned c DN A fragment was cloned for orientation determination.
  • the 0138a06 clone contains a full-length cDNA of 2533bp (as shown in Seq ID NO: 1). There is a 428bp open reading frame (0RF) from 801bp to 1229bp. It encodes a new protein (such as Seq ID NO: 2).
  • This clone pBS-0138a06 was named the encoded protein.
  • Heliosome S7 protein 16 Implemented column 2: RT-PCR method was used to expand the base encoding human ribosomal S7 protein 16
  • RNA of brain cells was used as a template, and oligo-dT was used as a primer to perform reverse transcription reaction to synthesize cDNA. After purification with Qiagene kit, the primers were used for PCR amplification:
  • Primerl 5'- GACAACAGAAATTTCAGTTATTAT -3 "(SEQ ID N0: 3)
  • Primer2 f- TATGTATAAGGTAAAGTATATTTA _3 ⁇ (SEQ ID NO: 4)
  • Primerl is a forward sequence starting from the 1st lbp at the 5 ′ end of SEQ ID NO: 1;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Conditions for the amplification reaction 50 mmol / L t (Cl, 10 cryptool / L Tris-CI, (pH 8.5), 1.5 mmol / L MgCI 2 , 200 ⁇ mol / L dNTP in a reaction volume of 50 ⁇ 1 , lOpmol primer, 1U of Taq DNA polymerase (product of Clomech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Eimer) for 25 cycles under the following conditions: 94. C 30sec; 55 ° C 30sec; 72 ° C 2min. Set ⁇ -act in as a positive control and template blank as a negative control at the same time during RT-PCR.
  • This method involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue is homogenized with 4 guanidine cyanocyanate-25m 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 was synthesized by electrophoresis on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7. Q)-5 mM sodium acetate-1 miM EDTA-2.2 M formaldehyde. It was then transferred to a nitrocellulose membrane.
  • the DNA probe used was the sequence of the human ribosome S7 protein 16 coding region (801bp to 1229bp) amplified by PCR shown in FIG. 1.
  • a 32P-labeled probe (approximately 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 (> C overnight in a solution containing 50 3 ⁇ 4 formamide-25 m ⁇ , ⁇ ( ⁇ 7.4) -5 SSC-5 Denhardt's solution and 200 yg / ml salmon sperm DNA. After hybridization, the filter was washed in 1 x SSC-0.13 ⁇ 4SDS for 30 min. Then, it was analyzed and quantified by Phosphor Imager.
  • Example 4 In vitro expression, isolation and purification of recombinant human ribosomal S7 protein 16
  • Primer3 5 *-CATGCTAGCATGACTAGAACTAAAACAGTAAAT -3 '(Seq ID No: 5)
  • Primer4 5'- CATGGATCCCTATTGTAACGTTGTTAGTAGTTC -3 "(Seq ID No: 6)
  • the 5' ends of these two primers contain Nhel and BamHI restriction sites, respectively. 5 'end of target gene
  • Nhe I and BamH I were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligation product was transformed into coliform bacteria DH5 CC using the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 30 g / ml), positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0138a06) with the correct sequence was selected, and the recombinant plasmid was transformed into E.
  • coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method.
  • host strain BL21 P ET-0138a06
  • IPTG was added to a final concentration of lmmol / L , Continue to cultivate for 5 hours.
  • the bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. Chromatography was performed using an affinity chromatography column His. Bind Quick Cartridge! Novagen company product capable of binding to 6 histidines (6His-Tag).
  • a purified g protein of human ribosomal S7 protein 16 was obtained. After SDS-PAGE electrophoresis, a single band was obtained at 16 kDa (Fig. 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by Edams hydrolysis method. The N-terminal 15 amino acid residues shown in NO: 2 are identical.
  • Example 5 Production of anti-human ribosomal S7 protein 16 antibodies
  • a peptide synthesizer (product of PE company) was used to combine the peptides specific for the human nucleus S7 protein 16: NH2-Me t-Thr-Arg-Thr-Ly s-Thr-Va 1-Asn-Leu-As p -Asn-11 e-Lys-Pro-G 1 n- C00H (SEQ ID NO: 7).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • the 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 from different tissues of royal or pathological tissues. and s columns identified polynucleotide sequences homologous to the polynucleotide carrier detected if it contains the present invention, further into a ho This probe is used to detect whether the polynucleotide sequence of the present invention or its homologous polynucleotide sequence is abnormally expressed in cells of normal tissue or pathological tissue.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern imprinting, Northern blotting, and copying methods. They all use the same steps to immobilize the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing 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.
  • the probe is large :: preferably 18-50 nucleotides
  • preliminary selection probes Those that meet the above conditions can be used as preliminary selection probes, and then further computer sequence analysis, including the preliminary selection ⁇ its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and Compare the homology of the regions with each other. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used generally
  • Probe 1 (probel), which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41 t):
  • probe2 which belongs to the second class of probes, and belongs to the gene fragment of SEQ ID NO: 1 Or its complementary mutant substitution sequence (41Nt):
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membrane nitric acid membrane
  • Pre-hybridization Place the sample in a plastic bag, add eight 3-1 Omg pre-hybridization solution (lOxDenhardf s; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA).), Seal the bag, and shake at 68 ° C in a water bath 2 hours.
  • 3-1 Omg pre-hybridization solution lOxDenhardf s; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA).
  • probe 1 can be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues.
  • Gene microarrays or DNA microarrays are new technologies currently being developed by many national laboratories and large pharmaceutical companies. They refer to the orderly and high-density arrangement of a large number of target gene fragments in a glass. And tritium, and then use fluorescence detection and computer software to compare and analyze the data, so as to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as a target DNA two-gene chip technology to study the function of new genes with two high-throughputs: find and screen for tissue specificity New genes, especially those related to diseases such as tumors; Diagnosis of diseases, such as hereditary diseases. The specific method steps have been reported in the literature.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotides of the present invention. They were respectively amplified by PCR. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and spotted on a sloped glass medium using a Cartesian 7500 spotter (purchased from Cartesian, USA) between the points. The distance is 280 ⁇ ⁇ ⁇ . The spotted slides were hydrated, dried, and cross-linked in a UV cross-linker. After elution, the slides were fixed to fix the DNA on the glass slides to prepare chips. The specific method steps have been variously reported in the literature. The post-spot processing steps of this embodiment are:
  • niRNA was extracted from human tissues and specific tissues (or stimulated cell lines) in one step, and mRNA was purified using Oligotex mRNA Midi Kit (purchased from QiaGen).
  • Cy3dUTP (5-Amino-propargy 1-2 '-deoxyur id ine 5 -triphate coupled to Cy3 fluorescent dye, purchased from Amersham Phamacia Biotech) was used to label the niRNA of human mixed tissue, and the fluorescent reagent CySdUTP (5-Amino-propargy 1- 2- -deoxyur id ine 5'-tr iphate coupled to Cy5 fluorescent dye, from Amersham Phamacia Biotech company) marker specific tissue (or stimulated cell line) niRNA, and probes were prepared after purification.
  • Cy3dUTP (5-Amino-propargy 1-2 '-deoxyur id ine 5 -triphate coupled to Cy3 fluorescent dye, purchased from Amersham Phamacia Bio
  • the above specific tissues are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, L02 cell line stimulated by arsenic for 1 hour, L02 cell line stimulated by arsenic for 6 hours prostate, heart, lung cancer, fetal bladder, fetal small intestine, fetal large intestine, thymus, fetal muscle, fetal liver, fetal kidney, fetal spleen, fetal brain Fetal lung and fetal heart.
  • polypeptides of the present invention 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.
  • Ribosomal protein S7 is one of the proteins in the small subunit of the ribosome: it plays a very important role in the correct tRNA selection in the initial stages of protein synthesis. The S7 protein can select the correct tRNA to bind to small ribosomal subunits to avoid mismatches, which is directly related to the stability of the passage of the organism. And the loss of ribosomal protein will slow down the growth of cells.
  • the abnormal expression of the specific S7 family protein motif will cause the dysfunction of the polypeptide containing the S7 family protein mot if of the present invention, resulting in the mistranslation of mRNA and the generation of related diseases such as tumors, embryonic development disorders, Growth and development disorders.
  • the abnormal expression of the human ribosomal S7 protein 16 of the present invention will produce various diseases, especially embryonic developmental disorders, growth and development disorders, various tumors, and inflammations. These diseases include, but are not limited to:
  • Embryonic developmental disorders congenital 'mainstream births, cleft palate, facial oblique fissures, limb absentness, limb differentiation disorders, gastrointestinal atresia or stenosis, diarrhea, atelectasis, polycystic kidney, ectopic kidney, double ureter, Recessive, congenital inguinal hernia, double uterus, vaginal atresia, hypospadias, hermaphroditism, atrial septal defect, ventricular septal defect, arterial stenosis, arterial duct occlusion, neural tube defect, congenital hydrocephalus, iris Defects, congenital cataract, congenital glaucoma or cataract, congenital deafness
  • Growth and development disorders mental retardation, cerebral palsy, brain development disorders, mental retardation, familial cerebral nucleus development syndrome, strabismus, skin, fat, and muscular dysplasia disorders such as congenital skin relaxation, ⁇ Oldness, 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, cholecystoma, colon cancer, thymic tumor, nasal cavity and sinus tumor, nose Pharyngeal cancer, Laryngeal cancer, Tracheal tumor, Pleural mesothelioma, Fibroid, Fibrosarcoma, Lipoma, Liposarcoma, Leiomyoma
  • the abnormal expression of the human ribosomal S7 protein 16 of the present invention will also produce certain hereditary, hematological and immune system diseases.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human ribosomal S7 protein 16.
  • Agonists increase human ribosomal S7 protein 16 to stimulate biological functions such as cell proliferation, while antagonists block 11 to treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or formulations expressing human ribosomal S7 protein 16 are cultured with labeled human ribosome S7 protein 16. The ability of a given drug to increase or block this interaction is then determined.
  • Antagonists of human ribosomal S7 protein 16 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human ribosomal protein 16 can bind to human ribosomal S7 protein 16 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions.
  • human ribosomal S7 protein 16 When screening compounds as antagonists, human ribosomal S7 protein 16 can be added to bioanalytical assays. Whether a compound is an antagonist can be determined by measuring the effect of the compound on the interaction between human ribosome S7 protein 16 and its receptor . In the same manner as described above for screening compounds, analogues of receptor deletions that act as antagonists can be screened. Polynucleotides capable of binding to human ribosomal S7 protein 16 can be obtained by screening random peptides composed of various possible amino acids bound to solids. Generally, human nuclear ⁇ S7 protein 16 should be screened.
  • the present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against the human ribosomal S7 protein 16 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human ribosomal S7 protein 16 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant. Wait.
  • Techniques for preparing monoclonal antibodies to human ribosomal S7 protein 16 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human B-cell hybridoma technology, EBV -Hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions to non-human variable regions can be produced using existing techniques (Morrison et ai, PNAS, 1985, 81: 6851). 0 Existing techniques for producing single-chain antibodies (US Pat No. .4946778). Single-chain antibodies against human ribosomal S7 protein 16 can be produced using two.
  • Antibodies against human ribosomal S7 protein 16 can be used in immunohistochemical techniques to detect human ribosomal S7 protein 16 in biopsy specimens.
  • Monoclonals that bind to human ribosomal S7 protein 16 can also be labeled with radioisotopes and injected into the body to track their location and distribution.
  • This radioactive: bio-labeled 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 but in situ body.
  • human ribosomal S7 protein 16 high-affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is an S thiol crosslinker such as SPDP, which attacks the amino group of an antibody and binds the toxin to the antibody through the exchange of disulfide bonds.
  • SPDP S thiol crosslinker
  • This hybrid antibody can be used to kill human ribosome S7 protein 16 positive cells .
  • the antibodies in the present invention can be used to treat or predict diseases related to human ribosomal S7 protein 16.
  • Administration of an appropriate dose of antibody can stimulate or block the production or activity of human ribosomal S7 protein 16.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human sugar S7 protein 16 levels. These tests are well known in the art and include FISH: determination and radioimmunoassay.
  • FISH determination and radioimmunoassay.
  • the level of human ribosomal S7 protein 16 detected in the test can be used to explain the importance of human ribosome S7 protein 16 in various diseases and to diagnose human ribosome S7 protein 16 and other diseases.
  • the peptides of the present invention can also be used as spectral spectra.
  • Mi-- Polypeptides can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, preferably mass spectrometry analysis.
  • the polynucleotide encoding human ribosomal S7 protein 16 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 ribosomal S7 protein 16.
  • Recombinant gene therapy vectors can be designed to express mutated human ribosome S7 protein 16 to inhibit endogenous human ribosome S7 protein 16 activity.
  • a variant human ribosome S7 protein 16 may be a shortened human ribosome S7 protein 16 that lacks a signaling domain. Although it can bind to downstream substrates, it lacks signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of human ribosomal S7 protein 16.
  • Virus-derived expression vectors 1 ⁇ 2 retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc.
  • a recombinant polynucleotide encoding human ribosomal S7 protein 16 can be packaged into liposomes and transferred into cells.
  • Methods for introducing polynucleotides into tissues or cells include: directly injecting the polynucleotides into tissues in vivo; or introducing the polynucleotides into cells in vitro through a vector (such as a virus, phage, or plasmid), and then introducing the cells Transplanted into the body, etc.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit human ribosomal S7 protein 16 mRNA 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, such as the solid-phase phosphoramidite chemical synthesis method for oligonucleotide synthesis.
  • Antisense RNA molecules can be transcribed outside or in vivo by DNA sequences encoding the RNA.
  • This DNA sequence has been integrated downstream of the vector's RNA polymerase promoter.
  • it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphorothioate or peptide bonds instead of phosphodiester bonds.
  • Polynucleosides encoding human ribosomal S7 protein 16 can be used for the diagnosis of diseases related to human ribosome S7 protein 16.
  • the polynucleotide encoding human ribosomal S7 protein 16 can be used to detect the expression of human ribosome S7 protein 16 or the abnormal expression of human ribosome S7 protein 16 in a disease state.
  • the DNA sequence encoding human ribosomal S7 protein 16 can be used to hybridize biopsy specimens to determine the expression status of human ribosome S7 protein 16.
  • Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and so on. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • a part or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray (Microarray) or a DNA core (also known as a "base S core"). Differential expression of genes in tissues can be analyzed by tritium analysis and gene diagnosis .
  • Human ribosomal S7 protein 16 specific primers can be used for RNA-polymerase chain reaction (RT-PCR) in vitro amplification to detect human ribosomal S7 protein 16 transcription products.
  • Detection of mutations in the human ribosomal S7 protein 16 gene can also be used to diagnose human ribosome S7 protein 16-related diseases.
  • Human ribosome S7 protein 16 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human ribosome S7 protein 16 DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR, and in situ hybridization. In addition, mutations may affect the expression of proteins, so Northern blotting and Western blotting can be used to indirectly determine 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. According to this article, in order to associate these sequences with disease-related genes, the important first step is to locate these DNA sequences on the chromosome.
  • PCR primers (preferably 15-35bp) can be used to prepare sequences based on cDNA, which can be used to locate sequences on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.
  • the c D N A clone was subjected to fluorescence in situ hybridization (F I S H) with the metaphase chromosome.
  • F I S H fluorescence in situ hybridization
  • the chromosome localization can be accurately performed in a step A.
  • 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 differences in cDNA or genomic sequence between the affected and unaffected individuals need to be determined. If a mutation is observed in some or some patients, and the mutation is not observed in any normal eight body, then the mutation may be a disease of the disease: 1K is more diseased and not diseased, usually, : 'Step and first look for staining Structural changes in the body, such as deletions or translocations that are visible from the chromosomal level or detectable with c DNA sequence-based PCR.
  • the cDNA accurately mapped to the disease-related chromosomal region can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase Figure resolution and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human ribosomal S 7 protein 16 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human ribosomal S 7 protein 16 to be 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 ribosomale S7 humaine 16, 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 ribosomale S7 humaine 16.
PCT/CN2001/000528 2000-03-29 2001-03-26 Nouveau polypeptide, proteine ribosomale s7 humaine 16, et polynucleotide codant pour ce polypeptide WO2001073013A1 (fr)

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CN00115263.7 2000-03-29
CN 00115263 CN1315440A (zh) 2000-03-29 2000-03-29 一种新的多肽——人核糖体s7蛋白16和编码这种多肽的多核苷酸

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] 21 December 1999 (1999-12-21), Database accession no. AC006389 *
DATABASE GENBANK [online] 4 February 1998 (1998-02-04), Database accession no. AC002311 *
DATABASE GENBANK [online] 4 May 1999 (1999-05-04), Database accession no. AC004765 *
DATABASE GENBANK [online] 8 October 1997 (1997-10-08), Database accession no. AC002376 *

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