WO2001032862A1 - Nouveau polypeptide, proteine humaine 20 ribosome s4, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, proteine humaine 20 ribosome s4, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001032862A1
WO2001032862A1 PCT/CN2000/000401 CN0000401W WO0132862A1 WO 2001032862 A1 WO2001032862 A1 WO 2001032862A1 CN 0000401 W CN0000401 W CN 0000401W WO 0132862 A1 WO0132862 A1 WO 0132862A1
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polypeptide
polynucleotide
human
ribosomal protein
sequence
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PCT/CN2000/000401
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English (en)
Chinese (zh)
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Yumin Mao
Yi Xie
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Shanghai Bio Door Gene Technology Ltd.
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Priority to AU13786/01A priority Critical patent/AU1378601A/en
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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

  • a new polypeptide human S4 ribosomal protein 20, and a polynucleotide encoding this polypeptide
  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, human S4 ribosomal protein 20, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and the polypeptide.
  • 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 RNA 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 (Zhai Zhonghe Higher Education Press, Cell Biology, PP122-129).
  • the functional research of ribosomes has focused on ribosomal RNA. There are many rRNA domains that determine the 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 homologous proteins are also found in yeast, higher plants, and animals.
  • S4de Ua- 41 has two functional domains, functional domain 1 is an al fa helix structure, and functional domain 2 contains five parallel beta fold structures and three a lfa helix structures. Functional domain 2 is inserted into functional domain 1. Functional domain 2 is also homologous to the ETS functional domain of eukaryotic transcription factors.
  • the sequence of the secondary structure of S4 is al-aS-ctS-ct aS-pl-p?- ⁇ 3- ⁇ 6- ⁇ 4- ⁇ 5- ⁇ 7, where functional domain 1 is all a-helix (al-a2- a3-a7), functional domain 2 is ⁇ 4- ⁇ 5- ⁇ and ⁇ 2- ⁇ 3- ⁇ 6- ⁇ 4- ⁇ 5, and is inserted into functional domain 1.
  • functional domain 1 is all a-helix (al-a2- a3-a7)
  • functional domain 2 is ⁇ 4- ⁇ 5- ⁇ and ⁇ 2- ⁇ 3- ⁇ 6- ⁇ 4- ⁇ 5, and is inserted into functional domain 1.
  • the specific structure has also been clarified, and the literature is available for reference (The EMBO Journa l Vo l 17 Nol 6 pp4545-4558, 1998).
  • S4 is an important component of ribosomes. S4 of prokaryotes can bind to 16S rRNA, thus affecting translation Accuracy, high-level structure of 16S rRNA, etc. (Nowotny and Nierhaus, 1988; Allen and Noller, 1989) According to research, in the absence of other proteins, S4 can also interact with 16S rRNA.
  • S4 is also an RNA-binding protein. Like other ribosomal proteins, the S4 ribosomal protein of prokaryotes can bind to its own mRNA ( ⁇ ⁇ longitudinal daughter mRNA) to regulate its own expression. In addition to S4, it also encodes Sll, 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) 0
  • amino acid sequence of the binding site of S4 to rRNA and mRNA has no special homology, but the structural similarity of these two RMs has not been proven, and this aspect needs further study.
  • S4 plays a role in the assembly process of 30S and maintains the authenticity of translation.
  • the functions of the S4 ribosomal protein 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.
  • S4 is in eukaryotic cells It also has the same function;) S4 can also bind to its own mRNA, thereby regulating the expression of itself and other ribosomal proteins; (3) S4 can also promote the assembly of small subunits of the ribosome, the assembly process in small subunits It 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 transcription process of eukaryotes.
  • polypeptide of the present invention was inferred and identified as human S4 ribosomal protein 20 (HS4RP20).
  • 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 20.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding human S4 ribosomal protein 20.
  • Another object of the present invention is to provide a method for producing human S4 ribosomal protein 20. Another object of the present invention is to provide an antibody against the polypeptide of the present invention, human S4 ribosomal protein 20. 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 20.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities of human S4 ribosomal protein 20.
  • a novel isolated human S4 ribosomal protein 20 is provided.
  • the polypeptide is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID NO: 2, or a conservative variant polypeptide thereof, or Its active fragment, or its active derivative, analog.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • a polynucleotide encoding these isolated polypeptides, the polynucleotide comprising a nucleotide sequence having at least 70 nucleotides with a nucleotide sequence selected from the group consisting of % Identity: (a) a polynucleotide encoding the aforementioned human S4 ribosomal protein 20; (b) a polynucleotide complementary to the polynucleotide (a).
  • the polynucleotide encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 2.
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 117-671 in SEQ ID NO: 1; and (b) a sequence having 1-1187 in SEQ ID NO: 1 Sequence of bits.
  • Fig. 1 is a comparison diagram of the amino acid sequence homology of the inventors S4 ribosomal protein 20 and Schizosaccharomyces cerevisiae S4 ribosomal protein.
  • the upper sequence is human S4 ribosomal protein 20, and the lower sequence is Schizosaccharomyces cerevisiae S4 ribosomal protein.
  • Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+”.
  • FIG. 2 is a polyacrylamide gel electrophoresis diagram (SDS-PAGE) of human S4 ribosomal protein 20 isolated. 20kDa is the molecular weight of the protein. The arrow indicates the isolated protein band. Summary of the invention
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state. .
  • isolated human S4 ribosomal protein 20 means that human S4 ribosomal protein 20 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify human S4 ribosomal protein 20 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 20 peptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human S4 ribosomal protein 20, which basically consists of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention may be naturally purified products or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (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 human S4 ribosomal protein 20.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the human S4 ribosomal protein 20 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 (III) such A type in which a mature polypeptide is fused to another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or a UV), a polypeptide sequence in which an additional amino acid sequence is fused to a mature polypeptide (such as Leader sequences or secreted sequences or sequences used to purify this polypeptide or protease sequences)
  • such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes a nucleoside of SEQ ID NO: 1 Acid sequence.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 1187 bases in length and its open reading frame (117-671) encodes 184 amino acids. According to the amino acid sequence homology comparison, it was found that this polypeptide has 49% homology with S. ribozyme protein of S. cerevisiae. It can be deduced that the human S4 ribosomal protein 20 has similar structure and Features.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide that includes the polypeptide and a polynucleotide that includes additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • This polynucleotide variant can be a naturally occurring allelic variant or a non-naturally occurring variant.
  • These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity between the two sequences).
  • the invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Fi col l, 42 ° C, etc .; or (3) only between two sequences Hybridization occurs only when the identity is at least 95%, and more preferably 97%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • the invention also relates to nucleic acid fragments that hybridize to the sequences described above.
  • nucleic acid fragment contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nuclei. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding human S4 ribosomal protein 20.
  • 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 20 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDNA of interest is to isolate mR from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • mRNA extraction There are many mature techniques for mRNA extraction, and kits are also commercially available (Qiagene).
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When 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 20 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 usually 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 of human S4 ribosomal protein 20 gene expression.
  • a method using DNA technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-rapid cDNA end rapid amplification method
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be measured 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, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising a polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a human S4 ribosomal protein 20 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology. .
  • a polynucleotide sequence encoding human S4 ribosomal protein 20 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors expressed in bacteria (Rosenberg, et al.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • DM sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis.
  • promoters are: l ac or trp promoter of E.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and 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.
  • a polynucleotide encoding human S4 ribosomal protein 20 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as insect cells such as Fly S2 or Sf9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote, such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl 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.
  • polynucleotide sequence of the present invention can be used to express or produce recombinant human S4 ribosomal protein 20 (Sc ience, 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 outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid
  • the polypeptide of the present invention (human S4 ribosomal protein 20) 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 the protein's 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, diseases of the endocrine system, diseases of the nervous system, various types of inflammation, and the like.
  • 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:
  • 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 phagocytic deficiency immunodeficiency diseases, complement system defects; (3) primary immunodeficiency diseases accompanied by other diseases, such as Down syndrome, short limbs Dwarfism, hereditary transcobalamin II deficiency with hypogammaglobulinemia, biotin-dependent carboxylase deficiency, Dun Can syndrome, thymoma, chronic disease skin mucosal candidiasis, aplastic anemia, Di George Syndrome, Wiscot t-Aldr 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 immuno
  • Allergic diseases bronchial asthma, aspirin asthma;
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human S4 ribosomal protein 20.
  • Agonists enhance biological functions such as human S4 ribosomal protein 20 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 20 can be cultured with labeled human S4 ribosomal protein 20 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 20 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human S4 ribosomal protein 20 can bind to human S4 ribosomal protein 20 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide such that the polypeptide cannot perform biological functions.
  • human S4 ribosomal protein 20 can be added to a bioanalytical assay to determine whether the compound is an antagonist by measuring the effect of the compound on the interaction between human S4 ribosomal protein 20 and its receptor. .
  • Receptor deletions and analogs that function 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 20 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, 20 molecules of human S4 ribosomal protein should be labeled.
  • the present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against the human S4 ribosomal protein 20 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments and Fab expression libraries Fragment.
  • Polyclonal antibodies can be produced by injecting human S4 ribosomal protein 20 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 S4 ribosomal protein 20 include, but are not limited to, hybridoma technology (Kohler and Mil te i n. Nature, 1975, 256: 495-497), triple tumor technology, human beta- Cell hybridoma technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that combine human constant regions with non-human variable regions can be produced using existing techniques (Morri et al, PNAS, 1985, 81: 6851). And existing techniques for producing single-chain antibodies (US Pa t No. 4946778) can also be used to produce single chain antibodies against human S4 ribosomal protein 20.
  • Antibodies against human S4 ribosomal protein 20 can be used in immunohistochemical techniques to detect human S4 ribosomal protein 20 in biopsy specimens.
  • Monoclonal antibodies that bind to human S4 ribosomal protein 20 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 20 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 the antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human S4 ribosomal protein 20 positive cells .
  • the antibodies of the present invention can be used to treat or prevent diseases related to human S4 ribosomal protein 20.
  • Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human S4 ribosomal protein 20.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human S4 ribosomal protein 20 levels. These tests are well known in the art and include FI SH assays and radioimmunoassays. The levels of human S4 ribosomal protein 20 detected in the test can be used to explain the importance of human S4 ribosomal protein 20 in various diseases and to diagnose diseases in which human S4 ribosomal protein 20 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.
  • the polynucleotide encoding human S4 ribosomal protein 20 can also be used for a variety of therapeutic purposes. Gene therapy technology can be used to treat patients suffering from the lack of expression or abnormal / inactive expression of human S4 ribosomal protein 20. Cell proliferation, development, or metabolic abnormalities. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human S4 ribosomal protein 20 to inhibit endogenous human S4 ribosomal protein 20 activity.
  • a variant human S4 ribosomal protein 20 may be a shortened human S4 ribosomal protein 20 lacking a signaling functional domain, and although it can bind to a downstream substrate, it lacks signaling activity.
  • recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of human S4 ribosomal protein 20.
  • 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 20 into a cell.
  • Methods for constructing a recombinant viral vector carrying a polynucleotide encoding human S4 ribosomal protein 20 can be found in existing literature (Sambrook, etal.).
  • a recombinant polynucleotide encoding human S4 ribosomal protein 20 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 20 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 solid-phase phosphoramidite chemical synthesis to synthesize oligonucleotides.
  • 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 RM polymerase promoter of the vector.
  • it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphorothioate or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human S4 ribosomal protein 20 can be used for the diagnosis of diseases related to human S4 ribosomal protein 20.
  • the polynucleotide encoding human S4 ribosomal protein 20 can be used to detect the expression of human S4 ribosomal protein 20 or the abnormal expression of human S4 ribosomal protein 20 in a disease state.
  • the DNA sequence encoding human S4 ribosomal protein 20 can be used to hybridize biopsy specimens to determine the expression of human S4 ribosomal protein 20.
  • Hybridization techniques include Southern 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 immobilized as a probe on a microarray (M i croar ray) or DNA chip (also known as "gene chip") for differential expression analysis and gene diagnosis of genes in tissues.
  • Human S4 ribosomal protein 20 specific primers can also be used to detect the transcription products of human S4 ribosomal protein 20 by RNA-polymerase chain reaction (RT-PCR) in vitro amplification.
  • Human S4 ribosomal protein 20 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human S4 ribosomal protein 20 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 protein expression, so Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, the specific loci of each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) can be used to mark chromosome locations. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared based on the cDNA, and the sequences can be located on the chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells that contain the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention by a similar method, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendel ian Inheritance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine if genes and genes have been mapped to chromosomal regions Relationship between diseases.
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all of the affected individuals and the mutation is not observed in any normal individual, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients 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 20 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human S4 ribosomal protein 20 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. Examples
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Quik mRNA I solat ion K it (Qiegene product) Isolate poly (A) mRNA from total RNA.
  • 2ug poly (A) mRNA is reverse transcribed to form cDNA.
  • the Smart cDNA cloning kit purchased from Clontech
  • the bacteria formed a cDNA library.
  • Dye terminate cycle reaction ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0156H07 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • the sequence of the human S4 ribosomal protein 20 and the encoded protein sequence of the present invention were subjected to the Blast program (Basic local alignment search tool) [Altschul, SF et al. J. Mol. Biol. 1990; 215: 403-10] Perform homologous searches in databases such as Genbank and Swissport.
  • the gene with the highest homology to the human S4 ribosomal protein 20 of the present invention is a known Schizosaccharomyces cerevisiae S4 ribosomal protein, which encodes a protein with accession number Z99531 in Genbank.
  • the results of protein homology are shown in Figure 1. The two are highly homologous, with an identity of 49% and a similarity of 73%.
  • Example 3 Cloning of a gene encoding human S4 ribosomal protein 20 by RT-PCR
  • CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, the following primers were used for PCR amplification:
  • Primerl 5'-GCTTTCATTGGCCCAGGGTCCCA-3 '(SEQ ID NO: 3)
  • Priraer2 5'-CTTACAAAGCAGTTGGGGCATTT-3 '(SEQ ID NO:)
  • Priraerl is a forward sequence starting at the lbp of the 5th end of SEQ ID NO: 1;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification reaction conditions 50 ⁇ l of Kol, KCl, 10 mmol / L Tris-Cl, (pH8.5), 1.5ramol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol in a reaction volume of 50 ⁇ 1 Primer, 1U Taq DNA Polymerization Enzyme (C 1 om ech).
  • the reaction was performed on a PE9600 DM thermal cycler (Pe rki n-Elmer) for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72. C 2min.
  • RT-PCR set ⁇ -act in as a positive control and template blank as a negative control.
  • the amplified product was purified using a QIAGEN kit and ligated to a PCR vector using a TA cloning kit (Invitrogen).
  • the DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as l-1187bp shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of human S4 ribosomal protein 20 gene expression
  • This method involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 time volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ) And centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The obtained RM precipitate was washed with 70% ethanol, dried and dissolved in water.
  • Electrophoresis was performed on 20% ⁇ IM on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-Im EDTA- 2.2M formaldehyde. It was then transferred to a nitrocellulose membrane. Preparation cc- 32 P dATP with 32 P- DNA probe labeled by the random primer method.
  • the DNA probe used was the PCR-encoded human S4 ribosomal protein 20 coding region sequence (117bp to 671bp) shown in FIG.
  • a 32P-labeled probe (approximately 2 x 10 6 cptn / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM H 2 P0 4 (pH 7.4) -5 x SSC-5 x Denhardt's solution and 200 ⁇ g / ml salmon sperm DNA. After hybridization, the filter was washed in 1 x SSC-0.1% SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 5 In vitro expression, isolation and purification of recombinant human S4 ribosomal protein 20
  • Primer3 5'-CCCCATATGATGGTGCGGAAGCTTAAGTTCCAC-3 '(Seq ID No: 5)
  • Primer4 5,-CCCGGATCCCTAGGCTTCCAGATCGAAGTCATC- 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 reaction was performed using pBS-0156H07 plasmid containing the full-length target gene as a template.
  • PCR reaction conditions are: total volume 50 ⁇ 1
  • the pBS-0156H07 plasmid contained 10 pg, and the primers Primer-3 and Primer-4 were lpmol and Advantage polymerase Mix (Clontech) 1 ⁇ 1, respectively.
  • Cycle parameters 94 ° C 20s, 60. C 30s, 68. C 2 min, a total of 25 cycles.
  • Ndel and BamHI 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 DH5ct by the calcium chloride method. After being cultured on LB plates containing kanamycin (final concentration 30 g / ml) overnight, positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0156H07) with the correct sequence was selected. The recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method.
  • the host bacteria BL21 (pET-0156H07) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1 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, and chromatography was performed using an His. Bind Quick Cartridge (product of Novagen) which can bind to 6 histidines (6His-Tag). The purified human protein S4 ribosomal protein 20 was obtained.
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • rabbits were immunized with 4 mg of the hemocyanin-polypeptide complex plus complete Freund's adjuvant. After 15 days, the rabbit was immunized with hemocyanin polypeptide complex plus incomplete Freund's adjuvant once. ⁇
  • the titer of antibody in rabbit serum was measured by ELISA using a 15 g / ml bovine serum albumin peptide complex-coated titer plate. Protein A-Sepharose was used to isolate total IgG from antibody-positive rabbit sera.
  • the peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography. Immunoprecipitation demonstrated that the purified antibody specifically binds to human S4 ribosomal protein 20.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine humaine 20 ribosome S4, 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 humaine 20 ribosome S4.
PCT/CN2000/000401 1999-10-29 2000-10-30 Nouveau polypeptide, proteine humaine 20 ribosome s4, et polynucleotide codant pour ce polypeptide WO2001032862A1 (fr)

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AU13786/01A AU1378601A (en) 1999-10-29 2000-10-30 A novel polypeptide, a human s4 ribosome protein 20 and the polynucleotide encoding the polypeptide

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CN 99119917 CN1302817A (zh) 1999-10-29 1999-10-29 一种新的多肽－人s4核糖体蛋白20和编码这种多肽的多核苷酸
CN99119917.0 1999-10-29

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004018680A1 (fr) * 2002-07-15 2004-03-04 Institute Of Gene And Brain Science Procede pour cribler un antigene tumoral

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [online] 17 October 1995 (1995-10-17), Database accession no. Z55621/HS53D2F *
DATABASE EMBL [online] 17 October 1995 (1995-10-17), Database accession no. Z55622/HS53D2R *
DATABASE GENBANK [online] 30 September 1998 (1998-09-30), Database accession no. AC003693 *
GENOME RES., vol. 8, no. 11, November 1998 (1998-11-01), pages 1097 - 1108 *
NAT. GENET., vol. 6, no. 3, March 1994 (1994-03-01), pages 236 - 244 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004018680A1 (fr) * 2002-07-15 2004-03-04 Institute Of Gene And Brain Science Procede pour cribler un antigene tumoral

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