WO2001075057A2 - Nouveau polypeptide, proteine ribosomale s4 humaine 12, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, proteine ribosomale s4 humaine 12, et polynucleotide codant pour ce polypeptide Download PDF

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WO2001075057A2
WO2001075057A2 PCT/CN2001/000524 CN0100524W WO0175057A2 WO 2001075057 A2 WO2001075057 A2 WO 2001075057A2 CN 0100524 W CN0100524 W CN 0100524W WO 0175057 A2 WO0175057 A2 WO 0175057A2
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polypeptide
polynucleotide
human
ribosomal protein
sequence
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PCT/CN2001/000524
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Chinese (zh)
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WO2001075057A3 (fr
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Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
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Priority to AU73779/01A priority Critical patent/AU7377901A/en
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Publication of WO2001075057A3 publication Critical patent/WO2001075057A3/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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, namely human S4 ribosomal protein 12, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique
  • Ribosomes are organelles that synthesize proteins. Their only function is to synthesize polypeptide chains from amino acids in accordance with the instructions of mRM. 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. It has a diameter of 25 dishes and is mainly composed of proteins and RNA. Ribosomal RNA is called rRNA. The protein content is about 40% and RNA is about 60%.
  • Protein molecules are mainly distributed on the surface of ribosomes, while rRNA is located inside, and the two are bound together by non-covalent bonds.
  • Cell Biology Zhai Zhonghe Higher Education Press PP122-129 The function of ribosomes has focused on ribosomal RNA. There are many rRNA functional domains that determine different functions of ribosomes. (Annu Rev Biochem 1991; 60 191-227)
  • S4 is one of the most important ribosomal proteins. It is a binding protein of the small subunit 30S of prokaryotes. It also has homologous proteins in eukaryotes and performs the same biological functions. S4 is expressed in both bacteria and chloroplasts, and S4 and its homologs are also found in yeast, higher plants, and animals.
  • S4delta-41 has two Functional domains, functional domain 1 are all alfa spiral structures, functional domain 2 contains five parallel beta folding structures and three alfa spiral structures. Functional domain 2 is inserted into functional domain 1, which is also homologous to the ETS functional domain of a eukaryotic transcription factor.
  • the sequence of the secondary structure of S4 is ⁇ - ⁇ 2- ⁇ 3- ⁇ 4- ⁇ 5- ⁇ 1- ⁇ 2- ⁇ 3- ⁇ 6- ⁇ 4- ⁇ 5- ⁇ 7, where functional domain 1 is all ⁇ -helix ( ⁇ 1- ⁇ 2- ⁇ 3- ⁇ 7), functional domain 2 is ⁇ 4- ⁇ 5- ⁇ - ⁇ 2- ⁇ 3- ⁇ 6- ⁇ 4- ⁇ 4- ⁇ 5 and is inserted into functional domain 1.
  • the specific structure has also been clarified, and literature is available for reference. (The EMBO Journal Vol 17 Nol6 pp4545-4558 , 1998) ⁇
  • S4 is an important component of ribosomes. S4 of prokaryotes can bind to 16S rRNA, which affects the accuracy of translation, the advanced structure of 16S rRNA, and so on. (Nowotny and Nierhaus, 1988; Allen and Noller, 1989) According to research, S4 can also interact with 16SrRNA in the absence of other proteins. ,
  • S4 is also an RNA-binding protein. Like other ribosomal proteins, the S4 ribosomal protein of prokaryotes can bind to its own encoding of mMA (alpha operon mRNA) to regulate its own expression. In addition, it also encodes S11, S13, and L17, so S4 can regulate the expression of itself and other ribosomal proteins by binding to polycistronic mRNA. (Dean and Nomura, 1980; Thomas et al., 1987) (The EMBO Journal Vol 17 Nol6 pp4559-4571, 1998)
  • amino acid sequence of the binding site of S4 to rRNA and mRNA has no special homology, but the structural similarity of these two RNAs has not been proven, and this aspect needs further study.
  • S4 ribosomal proteins are as follows: (1) S4 can bind to 16S rRNA in prokaryotes, which affects the accuracy of translation, the advanced structure of 16S rRNA, and so on. It is speculated that S4 is in eukaryotic cells It also has the same function; (2) S4 can also bind to its own mRNA, thereby regulating the expression of itself and some other ribosomal proteins; (3) S4 can also promote the assembly of small subunits of the ribosome. The assembly process plays a role in maintaining the authenticity of translation; (4) S4 is also homologous with the ETS domain of eukaryotic transcription factors, so it is speculated that it can regulate the eukaryotic transcription process.
  • the human S4 ribosomal protein 12 protein plays an important role in regulating important functions of the body such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, so there has been a need in the art to identify more involved in these processes
  • the human S4 ribosomal protein 12 protein identifies the amino acid sequence of this protein. Isolation of the new human S4 ribosomal protein 12 protein encoding gene also provides a basis for the study to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic agents for disease 1 and it is therefore important to isolate its encoding MA. Disclosure of invention
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding human S4 ribosomal protein 12.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding human S4 ribosomal protein 12.
  • Another object of the present invention is to provide a method for producing human S4 ribosomal protein 12.
  • Another object of the present invention is to provide antibodies against the polypeptide of the present invention-human S4 ribosomal protein 12.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention-human S4 ribosomal protein 12.
  • Another object of the present invention is to provide a method for diagnosing and treating a disease associated with an abnormality of human S4 ribosomal protein 12.
  • the present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 927-1259 in SEQ ID NO: 1; and (b) a sequence having 1-1630 in SEQ ID NO: 1 Sequence of bits.
  • the present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of human S4 ribosomal protein 12 protein, which comprises using the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for in vitro detection of a disease or disease susceptibility associated with abnormal expression of human S 4 ribosomal protein 12 protein, which comprises detecting a mutation in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, or Detection of the amount or biological activity of a polypeptide of the invention in a biological sample.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of human S 4 ribosomal protein 12.
  • 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 a complete natural amino acid related to the protein molecule .
  • a “variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response in appropriate animals or cells and to bind to specific antibodies.
  • Antagonist refers to a protein that, when combined with human S4 ribosomal protein 12, causes a change in that 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 S4 ribosomal protein 12.
  • Antagonist refers to a molecule that, when combined with human S4 ribosomal protein 12, can block or regulate the biological or immunological activity of human S4 ribosomal protein 12.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that binds human S4 ribosomal protein 12.
  • Regular refers to a change in the function of human S4 ribosomal protein 12, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological, functional, or immune properties of human S4 ribosomal protein 12.
  • Those skilled in the art can purify human S4 ribosomal protein 12 using standard protein purification techniques. Basically Pure human S4 ribosomal protein 12 can generate a single main band on a non-reducing polyacrylamide gel. The purity of human S4 ribosomal protein 12 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 Nor thern blotting, 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 that the two sequences bind to each other as either specific or selective interactions.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Mad is on Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Clus ter method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). 0 The Cluster method checks all pairs The distances of each group 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 matching between sequence A and sequence B
  • the number of residues in sequence A-the number of spacer residues in sequence A-the number of spacer residues in sequence B can also be determined by the C l uster method or by methods known in the art such as Jotun He in Percent identity (He in J., (1990) Me thods in erazumo l ogy 183: 625-645).
  • Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitutions for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and? ⁇ It can specifically bind to the epitope of human S4 ribosomal protein 12.
  • 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 is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not part of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances in the natural state .
  • isolated human S4 ribosomal protein 12 refers to the human S4 ribosomal protein 12 group It does not contain other proteins, lipids, sugars or other substances naturally associated with it. Those skilled in the art can purify human S4 ribosomal protein 12 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 S4 ribosomal protein 12 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human S4 ribosomal protein 12, which is basically composed of SEQ ID NO: 1;
  • 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 can be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of human S4 ribosomal protein 12.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the human S4 ribosomal protein 12 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) such a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or (in) such One, wherein the mature polypeptide is fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) such a polypeptide sequence in which an additional amino acid sequence is fused into the mature polypeptide ( Such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protease sequence)
  • an additional amino acid sequence is fused into the mature polypeptide
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a full-length polynucleotide sequence of 1104 bases, and its open reading frame 927-1259 encodes 109 amino acids.
  • this polypeptide has a similar expression profile with human S4 ribosomal protein 20, and it can be deduced that the human S4 ribosomal protein 12 has similar functions to human S4 ribosomal protein 20.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DM can be single-stranded or double-stranded.
  • the DM can be a coding chain or a non-coding chain.
  • 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" is in the present invention It 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.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the present invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity, between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • "strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) Add a denaturant during hybridization, 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, most preferably at least 100 nucleotides. Nucleotides or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding human S4 ribosomal protein 12.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding the human S4 ribosomal protein 12 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 DM of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating 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. There are several mature techniques for fflRNA extraction. Kits are also commercially available (Qiagene).
  • CDM libraries are also commonly used (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology 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 loss of marker gene function; (3) determination of the level of human S4 ribosomal protein 12 transcripts; (4) ) Detection of protein products expressed by genes through immunological techniques or determination of 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, preferably within 1000 nucleotides.
  • the probe used here is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product expressed by the human S4 ribosomal protein 12 gene.
  • ELISA enzyme-linked immunosorbent assay
  • a method using PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-cDM terminal rapid amplification method
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein.
  • the amplified DM / RNA fragment can be isolated and purified by conventional methods such as 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 cDM sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell that is genetically engineered using the vector of the present invention or directly using a human S4 ribosomal protein 12 coding sequence, and recombinant technology A method for producing a polypeptide according to the invention.
  • a polynucleotide sequence encoding human S4 ribosomal protein 12 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 (Ros enberg, 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 incorporate a replication origin, a promoter, a marker gene, and translational regulatory elements.
  • Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human S4 ribosomal protein 12 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, earning synthesis technology, in vivo recombination technology, etc. (Sambroook, etal. Mo l ecu l ar C l on i ng, a Labora tory Manua l, co ld Harbor Harbora tory. New York, 1989 ).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct 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 for translation initiation, a transcription terminator, and the like. 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, polytumor enhancers on the late side of the origin of replication, and adenoviral enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • the vector can be transformed or transduced into a host cell to form a genetically engineered host cell containing the polynucleotide or a recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, Such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: E. coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as fly S2 or Sf 9; animal cells such as CH0, COS, or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence according to the present invention or a recombinant vector containing the DM sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the (3 ⁇ 4 (12 ) method, the steps used are well known in the art.
  • 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 lipid Plastid packaging, etc.
  • polynucleotide sequence of the present invention can be used to express or produce recombinant human S4 ribosomal protein 12 (Scence, 1984; 224: 1431). Generally, the following steps are taken:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • 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.
  • the polypeptide of the present invention (human S4 ribosomal protein 12) is an important ribosomal protein, which directly regulates the structure and assembly of small ribosomal subunits, the expression of itself and some other ribosomal proteins, and indirectly Regulates protein translation and expression, cell transcription and other life activities.
  • the polypeptide of the present invention can be used for the diagnosis and treatment of many diseases, such as malignant tumors, immune diseases, endocrine system diseases, nervous system diseases and various inflammations.
  • FIG. 1 is a comparison diagram of gene chip expression profiles of S4 ribosomal protein 12 and human S4 ribosomal protein 20 of the present invention.
  • the upper graph is a graph of the expression profile of human S4 ribosomal protein 12, and the lower graph is the graph of the expression profile of human S4 ribosomal protein 20.
  • 1 indicates fetal kidney
  • 2 indicates fetal large intestine
  • 3 indicates fetal small intestine
  • 4 indicates fetal muscle
  • 5 indicates fetal brain
  • 6 indicates fetal bladder
  • 7 indicates non-starved L02
  • 8 indicates L02 +, lhr, As 3+
  • 9 indicates ECV304 PMA-
  • 10 means ECV304 PMA +
  • 11 means fetal liver
  • 12 means normal liver
  • 13 means thyroid
  • 14 means skin
  • 15 means fetal lung
  • 16 means lung
  • 17 means lung cancer
  • 18 means fetal spleen
  • 19 means spleen
  • 20 Indicates prostate
  • 21 indicates fetal heart
  • 22 indicates heart
  • 23 indicates muscle
  • 24 indicates testis
  • 25 indicates fetal thymus
  • 26 indicates thymus.
  • Figure 1 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of human S4 ribosomal protein 12 isolated.
  • 121 cDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band. The best way to implement the invention
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. Separation Quik mRNA Isolat ion Ki t (Qiegene Co.) from the total MA in poly (A) mRNA 0 2ug poly (A) mRNA by reverse transcription formed cDM.
  • the Smart cDNA cloning kit purchased from Clontech was used to insert the cDNA fragments into the multiple cloning site of pBSK (+) vector (Clontech) to transform DH5a. The bacteria formed a cDNA library.
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • CDNA The sequence was compared with the existing public DNA sequence database (Genebank), and the cDNA sequence of one of the clones 0125e03 was found to be new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • 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'- AATGATAGGGCTGTTTCGCTACAG-3 '(SEQ ID NO: 3)
  • Primer2 5'- GCAGCCCCGGCCCGGGAGACCGCT-3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Primer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.
  • Amplification conditions 50 ⁇ l of KC1, 10 ⁇ l / L of Tris-
  • RNA was synthesized by electrophoresis on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (H7.0)-5 mM sodium acetate-1 mM EDTA-2.2M formaldehyde. It was then transferred to a nitrocellulose membrane.
  • the DNA probe used was the PCR amplified human S4 ribosomal protein 12 coding region sequence (927bp to 1259bp) shown in FIG. 1.
  • Primer3 5'- CATGCTAGCATGCAGATTTTTTTTTTTTTTTTTTTTTT-3 '(Seq ID No: 5)
  • Primer4 5'- CATGGATCCTCAAATGAACTTTCTGAAACCCAT- 3' (Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and BamHI restriction sites, respectively.
  • the coding sequences for the 5 'and 3' ends of the gene of interest are followed, respectively.
  • the Ndel and BamHI restriction sites correspond to the selectivity within the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Digestion site.
  • PCR was performed using the pBS-0125e03 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1, containing 10 pg of pBS- 0125e03 plasmid, Primer- 3 and Primer- 4 points, and I] lOpmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1.
  • Cycle parameters 94 ° C 20s, 60 ° C 30s, 68. C 2 min, a total of 25 cycles.
  • Ndel and BaraHI were used to double-digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligation product was transformed into E. coli DH5CC using the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 30 ⁇ g / ml), positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0125e03) with a correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method.
  • the host bacteria BL21 (pET-0125e03) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1 to make ol / 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.
  • the purified human protein S4 ribosomal protein 12 was obtained.
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern 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), so that the hybridization background is reduced and only strong specific signals are retained.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • oligonucleotide fragments 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 size of the probe ranges from 18 to 50 nucleotides; 2, GC content is 3-70%, 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, the primary probe should not be used;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 which belongs to the second type of probe, is equivalent to the replacement mutation sequence (41Nt) of the gene fragment or its complementary fragment of SEQ ID NO: 1:
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membrane nitrocellulose membrane
  • Gene chip or DNA microarray is a new technology that many national laboratories and large pharmaceutical companies are currently developing and developing. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of fast, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • the specific method steps have been reported in the literature. For example, refer to the literature DeRi si, JL, Lyer, V. Brown, P. 0. (1997) Science 278, 680-686. And the literature Hel le, RA, Schema , M., Cha i, A., Sha lom, D., (1997) PNAS 94: 2150-2155.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were respectively amplified by PCR. After purification, the amplified product was adjusted to a concentration of about 500 ng / ul, and spotted on a sloped medium using a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance from the point is 280 ⁇ ⁇ . The spotted slides were hydrated, dried, and cross-linked in a purple diplomatic coupling instrument. After elution, the DNA was fixed on a glass slide to prepare a chip. The specific method steps have been reported in the literature in various ways. The post-spot processing steps of this embodiment are:
  • Cy5dUTP (5- Amino-propargy 1-2'- deoxyur idine 5'-tr iphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech company) labeled specific tissues (or stimulated cell lines) mMA of the body, and purified probes.
  • Cy5 fluorescent dye purchased from Amersham Phamacia Biotech company
  • Probes from the above two types of tissues were hybridized with the chip in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, washed with a washing solution (lx SSC, 0.2% SDS) at room temperature and scanned with ScanArray 3000
  • the instrument purchased from General Scanning Company, USA was used for scanning.
  • the scanned images were analyzed and processed with Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, L02 cell line stimulated by arsenic for 1 hour, L02 cell line stimulated by arsenic for 6 hours prostate, heart, lung cancer, fetal bladder, fetal small intestine, fetal large intestine, fetal thymus, fetal muscle, fetal liver, fetal kidney, fetal spleen, fetal brain, Fetal lung and fetal heart.
  • the polypeptide of the present invention can be used to treat human malignant tumors, including but not limited to gastric cancer, liver cancer, colorectal cancer, breast cancer, lung cancer, prostate cancer, cervical cancer, pancreatic cancer, and esophageal cancer.
  • the polypeptide of the present invention is also an immunomodulator, and has an immune promoting or immunosuppressing effect.
  • the polypeptide of the present invention can be used for the treatment of some diseases, including non-response of the immune response, or abnormal immune response, or ineffective host defense. Examples are as follows:
  • Human major histocompatibility antigen-related diseases rheumatoid arthritis, chronic active hepatitis, primary dryness syndrome, acute anterior uveitis, arthritis after gonococcal infection, ankylosing spondylitis, hemochromatosis , Immune complex glomerulonephritis, myocarditis after gonococcal infection; Autoimmune diseases: systemic lupus erythematosus, rheumatoid arthritis, scleroderma, polymyositis, xerostomia, nodular polyarteritis, autoimmune gastritis, insulin autoimmune syndrome, autoimmune Immune thyroid disease, autoimmune heart disease;
  • Primary immunodeficiency disease (1) Primary specific immunodeficiency disease such as antibody-based primary specific immunodeficiency disease, combined immunodeficiency disease, primary specific immunodeficiency disease with other characteristics; (2) primary non-specific immunodeficiency diseases such as immunodeficiency diseases that lack phagocytic cells, complement system defects; (3) primary immunodeficiency diseases with other diseases such as Down syndrome, short-limb dwarfism, Hereditary transcobalamin II deficiency with hypogammaglobulinemia, biotin-dependent carboxylase deficiency, Dun Can syndrome, thymoma, chronic disease skin mucosal candidiasis, aplastic anemia, D i George syndrome , Wi s co t t- A l dr i ch syndrome, immunodeficiency disease with ataxia capillary dilatation;
  • Primary specific immunodeficiency disease such as antibody-based primary specific immunodeficiency disease, combined immunodeficiency disease, primary specific immunodefic
  • Allergic diseases bronchial asthma, aspirin asthma;
  • 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 mitochondrial disease, metabolic disorders related to energy and material metabolism, and growth and development disorders. Diseases, congenital malformations, certain tumors, certain hereditary, hematological and immune system diseases, etc.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human S4 ribosomal protein 12.
  • Agonists enhance biological functions such as human S4 ribosomal protein 12 to stimulate cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing human S4 ribosomal protein 12 can be cultured with labeled human S4 ribosomal protein 12 in the presence of drugs. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human S4 ribosomal protein 12 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human S4 ribosomal protein 12 can bind to human S4 ribosomal protein 12 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 S4 ribosomal protein 12 can be added to bioanalytical assays to determine whether the compound is an antagonist by measuring the effect of the compound on the interaction between human S4 ribosomal protein 12 and its receptor. .
  • Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to human S4 ribosomal protein 12 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, the 12 molecules of human S4 ribosomal protein should generally be labeled.
  • the present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against the human S4 ribosomal protein 12 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 S4 ribosomal protein 12 directly into immunized animals (such as rabbits, mice, rats, etc.). Various 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 S4 ribosomal protein 12 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridization Tumor technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions to non-human variable regions can be produced using existing techniques (Morris on et al, PNAS, 1985, 81: 6851).
  • the existing technology for producing single chain antibodies U.S. Pat No. 4946778, can also be used to produce single chain antibodies against human S4 ribosomal protein 12.
  • Antibodies against human S4 ribosomal protein 12 can be used in immunohistochemical techniques to detect human S4 ribosomal protein 12 in biopsy specimens.
  • Monoclonal antibodies that bind to human S4 ribosomal protein 12 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • human S4 ribosomal protein 12 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 sulfhydryl crosslinker such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human S4 ribosomal protein 12 positive cells .
  • the antibodies of the present invention can be used to treat or prevent diseases related to human S4 ribosomal protein 12. Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human S4 ribosomal protein 12.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human S4 ribosomal protein 12 levels. These tests are well known in the art and include FISH assays and radioimmunoassays. The levels of human S4 ribosomal protein 12 detected in the test can be used to explain the importance of human S4 ribosomal protein 12 in various diseases and to diagnose diseases in which human S4 ribosomal protein 12 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 analysis.
  • Polynucleotides encoding human S4 ribosomal protein 12 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 S4 ribosomal protein 12.
  • Recombinant gene therapy vectors can be designed to express mutated human S4 ribosomal protein 12 to inhibit endogenous human S4 ribosomal protein 12 activity.
  • a variant human S4 ribosomal protein 12 may be a shortened human S4 ribosomal protein 12 lacking a signaling functional domain. Although it can bind to a downstream substrate, it lacks signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of human S4 ribosomal protein 12.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus and the like can be used to transfer a polynucleotide encoding human S4 ribosomal protein 12 into cells.
  • Methods for constructing a recombinant viral vector carrying a polynucleotide encoding human S4 ribosomal protein 12 can be found in existing literature (Sambrook, eta l.).
  • a recombinant polynucleotide encoding human S4 ribosomal protein 12 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 S4 ribosomal protein 12 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 RM or DM synthesis techniques, such as the solid-phase phosphoramidite synthesis method for oligonucleotide synthesis.
  • 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 linkage between ribonucleosides using phosphorothioate or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human S4 ribosomal protein 12 can be used for the diagnosis of diseases related to human S4 ribosomal protein 12.
  • the polynucleotide encoding human S4 ribosomal protein 12 can be used to detect the expression of human S4 ribosomal protein 12 or the abnormal expression of human S4 ribosomal protein 12 in a disease state.
  • the DM sequence encoding human S4 ribosomal protein 12 can be used to hybridize biopsy specimens to determine the expression of human S4 ribosomal protein 12.
  • Hybridization techniques include Sou thern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • Part or all of the polynucleotides of the present invention can be used as probes to be fixed on a micro array (Mi croar ray) or a DNA chip (also known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • a micro array Mo croar ray
  • a DNA chip also known as a "gene chip”
  • Human S4 ribosomal protein 12 specific primers can be used for RNA-polymerase chain reaction (RT-PCR) in vitro amplification to detect human S4 ribosomal protein 12 transcription products.
  • Human S4 ribosomal protein 12 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human S4 ribosomal protein 12 DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, the mutation may affect the expression of the protein, so the Nor thern blotting and Western blotting can be used to indirectly determine whether there is a mutation in the gene.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for marking chromosome positions.
  • an important first step is to locate these Book A sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared according to cDM, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckus i ck, Mendel i an Inher i tance 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 sequences 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 staining Structural changes in the body, such as deletions or translocations that are visible from the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • 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 S4 ribosomal protein 12 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human S4 ribosomal protein 12 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 S4 humaine 12, et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la protéine ribosomale S4 humaine 12.
PCT/CN2001/000524 2000-03-29 2001-03-26 Nouveau polypeptide, proteine ribosomale s4 humaine 12, et polynucleotide codant pour ce polypeptide WO2001075057A2 (fr)

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CN00115295A CN1315455A (zh) 2000-03-29 2000-03-29 一种新的多肽——人s4核糖体蛋白12和编码这种多肽的多核苷酸
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4865970A (en) * 1986-02-28 1989-09-12 Hoffmann-La Roche Inc. Method of detecting ribosomal protein antibodies in systemic lupus erythematosus
WO2000028079A2 (fr) * 1998-11-09 2000-05-18 Gemini Genomics Ab Variation genetique associee a l'anemie aplasique, et applications diagnostiques et therapeutiques basees sur cette variation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4865970A (en) * 1986-02-28 1989-09-12 Hoffmann-La Roche Inc. Method of detecting ribosomal protein antibodies in systemic lupus erythematosus
WO2000028079A2 (fr) * 1998-11-09 2000-05-18 Gemini Genomics Ab Variation genetique associee a l'anemie aplasique, et applications diagnostiques et therapeutiques basees sur cette variation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [Online] 23 November 1999 LAWLOR S.: 'Human DNA sequence from clone 170F5 on chromosome Xq22.3-24. Contains an HMG1 (high-mobility group (nonhitone chromosomal) protein 1) pseudogene and ESTs, STSs and GSSs' Database accession no. AL035067.2 *
DECKERT G. ET AL.: 'The complete genome of the hyperthermophilic bacterium aquifex aeolicus' NATURE vol. 392, no. 6674, 1998, pages 353 - 358 *
IVENS A.C. ET AL.: 'A physical map of the leischmania major friedlin genome' GENOME RESEARCH vol. 8, no. 2, 1998, pages 135 - 145 *

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