WO2002002611A1 - Nouveaux polypeptides, domaine de repetition 12 de recepteurs peptidiques de la motilite du sperme et proteine ribosomale l22, et polynucleotides codant ces polypeptides - Google Patents

Nouveaux polypeptides, domaine de repetition 12 de recepteurs peptidiques de la motilite du sperme et proteine ribosomale l22, et polynucleotides codant ces polypeptides Download PDF

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Publication number
WO2002002611A1
WO2002002611A1 PCT/CN2001/000915 CN0100915W WO0202611A1 WO 2002002611 A1 WO2002002611 A1 WO 2002002611A1 CN 0100915 W CN0100915 W CN 0100915W WO 0202611 A1 WO0202611 A1 WO 0202611A1
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polypeptide
polynucleotide
sperm
repeat domain
ribosomal protein
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PCT/CN2001/000915
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English (en)
Chinese (zh)
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Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc.
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Priority to AU95370/01A priority Critical patent/AU9537001A/en
Publication of WO2002002611A1 publication Critical patent/WO2002002611A1/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

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide-ribosomal protein L22 and a sperm-activating peptide receptor repeating domain 12 and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide.
  • Ribosomal protein L22 is one of the proteins derived from the large ribosomal subunit. L22 binds to 23S rRNA. It belongs to the ribosomal protein family. The mammalian L17 protein is a member of this family. The C-terminal part of this family member has a conserved characteristic sequence template: [RKQN] -X (4)-[RH] -[GAS] - ⁇ -G- [KRQS] -x (9)-[HDN]-[LIVM] -x- [LIVMS] -x- [LIVM] (Otaka E., Hashimoto T., Mizuta L, Suzuki L, Protein Seq. Data Anal. 5: 301-313 (1993)).
  • Ribosomal protein L22 also plays an important role in the translation process. If this ribosome protein is missing or chemically modified, or its gene is mutated, it will affect the function of ribosomes and reduce the activity of peptide synthesis. Ribosomal protein L22 is very important for rRM to fold into a functional three-dimensional structure. In protein synthesis, a series of changes in the spatial conformation of the ribosome may occur. L22 may play a "fine-tuning" role in the conformation of the ribosome. The binding site may even play a catalytic role, and the L22 protein and rRNA function together.
  • the receptor for the sperm-activating peptide (a sea urchin egg peptide that stimulates sperm breathing) is a transmembrane glycoprotein containing approximately 500 amino acid residues (Dangott JJ, Jordan JE, Bel let R ⁇ , Garbers DL ; Proc Nat l. Acad. Sci. USA 86: 2128-2132 (1989)) Structurally, it consists of a large extracellular domain of about 450 residues followed by a transmembrane domain and a small 12 amino acid
  • the cytoplasmic domain is composed of an extracellular domain consisting of a 115 amino acid region of four repeats. Of the four repeats, 17 positions are absolutely conserved.
  • the sequence of the C-terminal part of the sperm active peptide family contains a conserved characteristic sequence template, which contains 8 of the 17 conserved residues: Gx (5)-Gx (2 ) -EX (6) -WGx (2) -Cx (3)-[FYW] -x (8)-C- x (3) -G (Freeman M., Ashkenas J., Rees DJ, Kings ley DM, Copeland NG, Jenkins NA, Krieger M., Proc. Nat l. Acad. Sci. USA 87: 8810- 8814 (1990) ).
  • the new polypeptide of the present invention has high homology and similarity with ribosome protein L22 and sperm-activating peptide receptor repeat domain family members in structure and function, and contains the two above-mentioned conserved characteristic sequence templates, so the polypeptide is considered to belong to The ribosomal protein L22 and sperm-activating peptide receptor repeat domain family, the polypeptide and its agonists, inhibitors, and antagonists can be used to treat various cancers and diseases such as atherosclerosis.
  • ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 protein play important roles in important functions in the body, and it is believed that a large number of proteins are involved in these regulatory processes, so the identification of more involved in these has been required in the art.
  • ribosomal protein L22 and sperm activating peptide receptor repeat domain 12 protein especially the amino acid sequence of this protein was identified. Isolation of the new ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 protein-encoding genes also provides a basis for research to determine its role in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding for DM.
  • 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 ribosomal protein L22 and a sperm-activating peptide receptor repeat domain 12.
  • Another object of the present invention is to provide a genetically engineered host cell comprising a polynucleotide encoding ribosomal protein L22 and a sperm activating peptide receptor repeat domain 12.
  • Another object of the present invention is to provide a method for producing ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12.
  • Another object of the present invention is to provide antibodies against the polypeptide-ribosomal protein L22 and the sperm-activating peptide receptor repeat domain 12 of the present invention.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide monoribosomal protein L22 and sperm activating peptide receptor repeat domain 12 of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of ribosomal protein L22 and sperm activating peptide receptor repeat domain 12. Mingzha
  • 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 468-797 in SEQ ID NO: 1; and (b) a sequence having 1-2848 in SEQ ID NO: 1 Sequence of bits.
  • the invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • a vector in particular an expression vector, containing the polynucleotide of the invention
  • a host cell genetically engineered with the vector including a transformed, transduced or transfected host cell
  • a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of ribosomal protein L22 and sperm-activating peptide receptor repeat domain 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 related to abnormal expression of ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 protein, which comprises detecting the polypeptide or a polynucleotide sequence encoding the same in a biological sample. Mutations, or the amount or biological activity of a polypeptide of the invention in a biological sample.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the preparation of polypeptides and / or polynucleotides of the present invention for the treatment of cancer, developmental or immune diseases or other diseases caused by abnormal expression of ribosomal protein L22 and sperm activating peptide receptor repeat domain 12. Use of medicine.
  • Figure 1 is a comparison of amino acid sequence homology of ribosome protein L22 and sperm-activating peptide receptor repeat domain 12 of the present invention at a total of 46 amino acids 49-94 and ribosome protein L22 and sperm-activating peptide receptor repeat domain characteristic domain homology .
  • the upper sequence is ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12
  • the lower sequence is Characteristic domains of ribosomal protein L22 and sperm-activating peptide receptor repeat domains.
  • Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+”. ;
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of isolated ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12. 12kDa is the molecular weight of the protein. The arrow indicates the isolated protein band.
  • 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 protein or polynucleotide “variant” refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. Variants can have "conservative" changes in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as the replacement of 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 means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that, when combined with ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12, can cause the protein to change, thereby regulating the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that binds to ribosomal protein L22 and the sperm-activating peptide receptor repeat domain.
  • Antagonist refers to when the ribosomal protein L22, sperm activating peptide receptor binding repeat domains I 2, an adjustable closure or ribosomal protein L22, sperm activating peptide receptor repeat domain 12 Biological activity Sexually or immunologically active molecules.
  • Antagonists and inhibitors can include proteins, nucleic acids, carbohydrates or any other molecule that can bind to ribosomal protein L22 and sperm activating peptide receptor repeat domain 12.
  • Regular refers to changes in the function of ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12, including increased or decreased protein activity, changes in binding characteristics, and ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 Of any other biological, functional or immune properties.
  • substantially pure means substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated.
  • Those skilled in the art can purify ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 using standard protein purification techniques.
  • the substantially pure ribosomal protein L22 and the sperm-activating peptide receptor repeat domain 12 can produce a single main band on a non-reducing polyacrylamide gel.
  • the purity of ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 polypeptides can be analyzed by amino acid sequence analysis.
  • 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 can 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 or Northern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of "fully homologous sequences to target sequences under conditions of reduced stringency. This does not mean that conditions of reduced stringency allow non-specific binding, Because of the reduced stringency conditions, the binding of two sequences to each other is a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madison Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Clus ter method (Higgins, D. G. and P. M. Sharp (1988) Gene 73: 237-244). The Cluster method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by:
  • Residues sequence a sequence number of residues in the interval ⁇ - ⁇ interval sequence of residues x
  • the percent identity between nucleic acid sequences can also be determined by the Clus ter method or by methods known in the art, such as Jotun Hein (Hein J., (1990) Methods in enzyraology 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.
  • negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; 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.
  • the "antisense strand” refers to a nucleic acid strand that is complementary to the “sense strand”.
  • Derivative refers to HFP or a chemical modification of its nucleic acid. 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?, Which can specifically bind to the epitope of ribosomal protein L22 and sperm-activating peptide receptor repeat domain 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 matter 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 animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist in the natural system.
  • Such a polynucleotide may be part of a vector, or such a polynucleotide or polypeptide may be part of a composition. Since the carrier or composition is not part of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state. .
  • isolated ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 means that ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 are substantially free of other proteins, lipids, Sugars or other substances.
  • Those skilled in the art can purify ribosomal protein L22 and sperm activating peptide receptor repeat domain 12 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of ribosomal protein L22 and sperm activating peptide receptor repeat domain 12 polypeptides can be analyzed by amino acid sequence analysis.
  • the present invention provides a novel polypeptide-ribosomal protein L22 and a sperm-activating peptide receptor repeat domain 12, which are basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the invention may be recombinant Peptides, natural polypeptides, synthetic polypeptides, preferably recombinant polypeptides.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. 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 ribosomal protein L22 and sperm activating peptide receptor repeat domain 12.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the ribosomal protein L22 and sperm activating peptide receptor repeat domain 12 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a type in which one or more amino acid residues are replaced 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 (II) such a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or (III) such One, in which the mature polypeptide is fused to another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV) such a polypeptide sequence in which the additional amino acid sequence is fused into the mature polypeptide ( Such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protein sequence).
  • 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 nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a CDM library of human fetal brain tissue. It contains a polynucleotide sequence of 2848 bases in total length and its open reading frame (468-797) encodes 109 amino acids.
  • This polypeptide has the characteristic sequences of the ribosome protein L22 and the sperm-activating peptide receptor repeat domain. It can be deduced that the ribosome protein L22 and the sperm-activating peptide receptor repeat domain 12 have the ribosome protein L22 and the sperm-activating peptide receptor repeat domain. Representing structure and function.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDM, genomic DM 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) Coded sequences) and non-coded sequences.
  • 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 (with at least two sequences between
  • 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 denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1 ° / »Ficol 1, 42 ° C, etc .; or (3) only between the two sequences Crosses occur at least 95% or more, and more preferably 97% or more.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 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 ribosomal protein L22 and sperm-activating peptide receptor repeat domain 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 ribosomal protein L22 and the sperm-activating peptide receptor repeat domain 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 DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of MA 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.
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spoon 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 can be screened from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DM or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; ( 3 ) determination of ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 The level of transcripts; (4) Detecting protein products expressed by genes by immunological techniques or by measuring biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein products of ribosomal protein L22 and sperm activating peptide receptor repeat domain 12 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA )Wait.
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA )Wait.
  • a method (Saiki, et al. Science 1985; 230: 1350-1354) using PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • 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 DM 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 cDM sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using ribosomal protein L22 and sperm activating peptide receptor repeat domain 12 coding sequence, and by recombinant technology A method for producing a polypeptide according to the invention.
  • a polynucleotide sequence encoding a ribosome protein L22 and a sperm-activating peptide receptor repeat domain 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, etal. Gene, 1987, 56: 125) expressed in bacteria; pMSXND expression vectors expressed in mammalian cells ( Lee and Nathans, J Bio Chem. 263: 3521, 1988) and baculovirus-derived vectors expressed in insect cells.
  • any plasmid and vector can be used to construct 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.
  • Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding ribosomal protein L22 and a sperm activating peptide receptor repeat domain 12 and suitable transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DM synthesis technology, and in vivo recombination technology (Sanibroook, et al. Mo l ecu l ar Cloning, a Labora tory Manua l, Coll Spring Harbor Labora tory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRM synthesis. Representative examples of these promoters are: the l ac or trp promoter of E.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Examples include 100 to 270 base pairs of the SV40 enhancer on the late side of the origin of replication, polyoma enhancers and adenovirus enhancers on the late side of the origin of replication.
  • 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 ribosomal protein L22 and a sperm-activating peptide receptor repeat domain 12 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a polynucleotide containing the polynucleotide or the recombinant vector.
  • Genetically engineered host cells refers to a prokaryotic cell, such as a bacterium Cells; or lower eukaryotic cells, 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 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.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 (Scence, 1984; 224: 1431). Generally there are the following steps:
  • polynucleotide or variant encoding human ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 of the present invention, or a recombinant expression vector containing the polynucleotide is used to transform or transduce a suitable host Cell
  • 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
  • polypeptides of the present invention can be directly used in the treatment of diseases, for example, they can be used to treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.
  • Ribosomal protein L22 is a large ribosomal protein. L22 binds to 23S rRNA and belongs to ribosomal protein. Family, ribosomal protein L22 also plays an important role in the translation process. If this ribosome protein is missing or chemically modified, or its gene is mutated, it will affect the function of ribosomes and reduce the activity of peptide synthesis. . Ribosomal protein L22 is important for rRNA folding into a functional three-dimensional structure. In protein synthesis, a series of changes in the spatial conformation of the ribosome may occur, and L22 may "fine-tune" the conformation of the ribosome. The C-terminal portion of the mammalian L22 protein family member has a conserved characteristic sequence template and is necessary for its activity.
  • the novel polypeptide of the present invention has a ribosome protein L22 functional domain in structure and function, contains a conserved characteristic sequence template, and has a functional domain of a sperm activating peptide receptor.
  • the functional domain of the sperm activating peptide receptor still exists In atherosclerotic pathological precipitated protein. It can be seen that the novel polypeptide of the present invention may also have the function of regulating atherosclerosis.
  • the abnormal expression of the ribosomal protein L22 functional domain and / or the sperm-activating peptide receptor functional domain of the novel polypeptide of the present invention will cause the function of the polypeptide containing the functional domain of the present invention to be abnormal, resulting in protein Synthetic abnormalities are closely related to the occurrence of pathological processes such as related material metabolic disorders, protein dysfunction, and tumors of related tissues, and produce related diseases such as atherosclerotic lesions.
  • ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 of the present invention will produce various diseases, especially atherosclerotic diseases, various tumors, embryonic development disorders, growth and development disorders, Inflammation, immune diseases, these diseases include but are not limited to:
  • Tumors of various tissues stomach cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioblastoma, nerve Fibroma, colon cancer, melanoma, bladder cancer, uterine cancer, endometrial cancer, thymic tumor, nasopharyngeal cancer, laryngeal cancer, tracheal tumor, fibroid, fibrosarcoma, lipoma, liposarcoma
  • Fetal developmental disorders congenital abortion, cleft palate, limb loss, limb differentiation disorder, atrial septal defect, neural tube defect, congenital hydrocephalus, congenital glaucoma or cataract, congenital deafness
  • Growth and development disorders mental retardation, brain development disorders, skin, fat and muscular dysplasia, bone and joint dysplasia, various metabolic deficiencies, stunting, dwarfism, Cushing syndrome, Sexual retardation
  • Inflammation chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, cerebrospinal multiple sclerosis, glomerulonephritis, myocarditis, cardiomyopathy, atherosclerosis, gastric ulcer, cervicitis, Various infectious inflammations
  • Immune diseases Systemic lupus erythematosus, rheumatoid arthritis, bronchial asthma, urticaria, specific dermatitis, post-infection myocarditis, scleroderma, myasthenia gravis, Guillain-Barre syndrome, common variable immunodeficiency disease , Primary B-lymphocyte immunodeficiency disease, Acquired immunodeficiency syndrome Abnormal expression of the ribosomal protein L22 and the sperm-activating peptide receptor repeat domain 12 of the present invention will also cause certain hereditary, hematological diseases, and the like.
  • 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 atherosclerosis, various tumors, embryonic developmental disorders, and developmental disorders. Diseases, inflammation, immune diseases, certain hereditary, blood diseases, etc.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12.
  • Agonists increase ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 can be cultured with labeled ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 in the presence of drugs. . The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of ribosomal protein L22 and sperm activating peptide receptor repeat domain 12 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 can bind to ribosome protein L22 and sperm-activating peptide receptor repeat domain 12 and eliminate their functions, or inhibit the production of the polypeptide, or with the polypeptide The active site binding prevents the polypeptide from performing biological functions.
  • ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 can be added to the bioanalytical assay. The effects of interactions between humans to determine whether a compound is an antagonist. 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 ribosomal protein L22 and sperm-activating peptide receptor repeat domain 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, 12 molecules of ribosomal protein L22 and sperm-activating peptide receptor repeat domain should be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against the ribosomal protein L22 and the sperm-activating peptide receptor repeat domain 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 immunized animals (such as rabbits, mice, rats, etc.) with ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 directly.
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including But it is not limited to Freund's adjuvant.
  • Preparation of ribosomal protein L22 and sperm activating peptide receptor weight Techniques for the monoclonal antibodies of the multidomain 12 include, but are not limited to, hybridoma technology (Kohler and Mistein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma technology, and EBV-hybridization. Tumor technology, etc.
  • Chimeric antibodies that bind human constant regions to non-human-derived variable regions can be produced using existing techniques (Morr ison et al, PNAS, 1985, 81: 6851).
  • the existing technology for producing single chain antibodies (US Pat No. 4946778) can also be used to produce single chain antibodies against ribosomal protein L22 and sperm activating peptide receptor repeat domain 12.
  • Antibodies against ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 can be used in immunohistochemical techniques to detect ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 in biopsy specimens.
  • Monoclonal antibodies that bind to ribosomal protein L22 and sperm-activating peptide receptor repeat domain 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 against a specific bead site in the body.
  • ribosomal protein L22 and sperm-activating peptide receptor repeat domain 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 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 ribosomal protein L22 and sperm-activating peptides.
  • Body repeat domain 12 positive cells are used.
  • the antibodies of the present invention can be used to treat or prevent diseases related to ribosomal protein L22 and sperm activating peptide receptor repeat domain 12.
  • Administration of appropriate doses of antibodies can stimulate or block the production or activity of ribosomal protein L22 and sperm activating peptide receptor repeat domain 12.
  • the invention also relates to a diagnostic test method for the quantitative and localized detection of ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 levels.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the levels of ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 detected in the test can be used to explain the importance of ribosome protein L22 and sperm-activating peptide receptor repeat domain 12 in various diseases and to diagnose ribose Diseases in which the body protein L22 and the sperm activating peptide receptor repeat domain 12 play a role.
  • 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 ribosomal protein L22 and sperm-activating peptide receptor repeat domain 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 absence or abnormal / inactive expression of ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 to suppress endogenous ribosomal protein L22 and sperm-activating peptide receptor repeats Domain 12 is active.
  • Gene therapy technology can be used to treat abnormal cell proliferation, development, or metabolism caused by the absence or abnormal / inactive expression of ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated ribosomal protein L
  • a variant ribosomal protein L22 and sperm-activating peptide receptor repeating domain I 2 may be shortened ribosome protein L22 and sperm-activating peptide receptor repeating domain 12 lacking a signaling function domain, although it may be related to downstream
  • the substrate binds but lacks signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of ribosomal protein L22 and sperm activating peptide receptor repeat domain 12.
  • Expression vectors derived from viruses such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc.
  • polynucleotides encoding ribosomal protein L22 and sperm activating peptide receptor repeat domain 12 can be used to transfer polynucleotides encoding ribosomal protein L22 and sperm activating peptide receptor repeat domain 12 to cells Inside.
  • Methods for constructing a recombinant viral vector carrying a polynucleotide encoding ribosomal protein L22 and a sperm-activating peptide receptor repeat domain 12 can be found in existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding ribosomal protein and sperm-activating peptide receptor repeat domain 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 ribosomal protein L22 and sperm activating peptide receptor repeat domain 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 MA for endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technology, such as solid-phase phosphate amide chemical synthesis to synthesize oligonucleotides.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RM. This DNA sequence has been integrated downstream of the vector's RNA polymerase promoter. In order to increase the stability of the nucleic acid molecule, it can be modified in various ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides should use phosphorothioate or peptide bonds instead of phosphodiester bonds.
  • Polynucleotides encoding ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 can be used to diagnose diseases related to ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12.
  • Polynucleotides encoding ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 can be used to detect the expression of ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 or activation of ribosomal protein L22 and sperm in disease states Aberrant Expression of Peptide Receptor Repeat Domain 12
  • DNA sequences encoding ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 can be used to hybridize biopsy specimens to determine the expression status of ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12.
  • 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. A part or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also known as a "gene chip") for analyzing the difference of genes in tissues. Heterologous analysis and genetic diagnosis. RNA-polymerase chain reaction (RT-PCR) amplification using ribosome protein L22 and sperm-activating peptide receptor repeat domain 12 specific primers can also detect the transcription of ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 product.
  • RT-PCR RNA-polymerase chain reaction
  • Ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 mutations can also be used to diagnose ribosome protein L22 and sperm-activating peptide receptor repeat domain 12-related diseases.
  • Ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 mutations include point mutations, translocations, deletions, recombinations, and others compared to normal wild-type ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 DNA sequences. Any exceptions etc. 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, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome positions. 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 cDNA, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DM to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and 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 differences in cDNA or genomic sequences between the affected and unaffected individuals need to be determined. If at A mutation is observed in some or all of the affected individuals, and the mutation is not observed in any normal individuals, then 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 using 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.
  • Ribosomal protein L22 and sperm activating peptide receptor repeat domain 12 are administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dose range of ribosomal protein L22 and sperm-activating peptide receptor repeat domain 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. Examples
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using Quik mRNA Isolat ion Kit (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA. Use Smart cDNA Cloning Kit (purchased from Clontech). The 0 fragment was inserted into the multicloning site of pBSK (+) vector (Clontech), and transformed into DH5 ⁇ . The bacteria formed a cDNA library.
  • the terminate cycle react ion sequencing kit (Perkin-Elmer) and the ABI 377 automatic sequencer (Perkin-Elmer) determined the sequences at the 5 'and 3' ends of all clones.
  • the determined cDNA sequence was compared with an existing public DM sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0595c02 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • the 0595c02 clone contains a full-length cDNA of 2848bp ('as shown by Seq ID N0: l), and has a 264bp open reading frame (0RF) from 468bp to 797bp, encoding a new protein (such as Seq ID NO: 2).
  • This clone pBS-0595c02 was named ribosomal protein L22 and sperm activating peptide receptor repeat domain 12.
  • the ribosomal protein L22 and the sperm-activating peptide receptor repeat domain 12 of the present invention and their encoded protein sequences were subjected to prof i le scan program (Basic local al ignment search tool) in GCG [Al tschul. SF et a. J. Mol. Biol. 1990; 215: 403-10], domain analysis was performed in a database such as prosit.
  • the ribosome protein L22 and the sperm-activating peptide receptor repeat domain 12 of the present invention are homologous with the domain ribosomal protein L22 and the sperm-activating peptide receptor repeat domain at 49-94, and the homology results are shown in FIG. 1.
  • the homology rate The value is 0.22 and the score is 9.97; the threshold value is 8.50.
  • Example 3 Cloning of genes encoding ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 by RT-PCR
  • CDNA was synthesized using fetal brain cell total MA as a template and ol igo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, the following primers were used for PCR amplification:
  • Pr imerl 5'- CATCCTGAGAACTGAAATTGATCGC- 3 '(SEQ ID NO: 3)
  • Pr imer2 5 '-ATAAAATTTTTGAATTTATGTTCAA-3' (SEQ ID NO: 4)
  • Primerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Pr imer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.
  • Conditions for the amplification reaction 50 ⁇ l of KCl, 10 mraol / L Tris-HCl pH 8.50, 1.5 mmol / L MgCl 2 , 20 ( ⁇ mol / L dNTP, l Opmol primer, 1U Taq DNA polymerase (product of Clontech).
  • the reaction was performed for 25 cycles on a PE9600 DM thermal cycler (Perkin-Elmer) under the following conditions: 94 ° C 30sec; 55. C 30sec; 72 ° C 2min. Set ⁇ -act in as a positive control and template blank as a negative control at the same time during RT-PCR.
  • the amplification product was purified using a QIAGEN kit and ligated to a pCR vector using a TA cloning kit (Invi trogen product). ).
  • the results of DM sequence analysis show that the DNA sequence of the PCR product is exactly the same as 1-2848bp shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12 gene expression
  • RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] 0
  • This method involves acid guanidinium thiocyanate-chloroform extraction. That is, the tissue was homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1), centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • RNA probe was the PCR amplified ribosomal protein L22 and the sperm activating peptide receptor repeat domain 12 coding region sequence (468bp to 797bp) shown in FIG.
  • a 32P-labeled probe (approximately 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 (pH7. 4)-5 x SSC-5 x Denhardt's solution and 200 g / ml salmon sperm DNA. After hybridization, place the filter at 1 x SSC-0. 1 ° /. Wash in 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 ribosomal protein L22 and sperm-activating peptide receptor repeat domain 12
  • Primer3 5'-CCCCATATGATGCTCTGTCACCTTCAAAGGATGG-3 '(Seq ID No: 5)
  • Primer4 5'- CCCAAGCTTCTTCAACATGCCGCTTCTGTTCTTC- 3' (Seq ID No: 6)
  • These two primers contain Ndel and Hindlll restriction sites, respectively.
  • the coding sequences of the 5 'and 3' ends of the target gene are followed, respectively.
  • the Ndel and Hindi II restriction sites correspond to the selection on the expression vector plasmid pET 28b (+) (Novagen, Cat. No. 69865. 3). Sex endonuclease site.
  • the PCR reaction was performed using the pBS-0595c02 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions are as follows: a total volume of 50 ⁇ 1 contains 10 pg of pBS-0595c02 plasmid, primers? ]: 11116]: -3 and? :: 111 ⁇ ]: -4 are 10 11101, 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 Hindlll 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 the colibacillus DH5 cx by the calcium chloride method. After being cultured overnight on an LB plate containing kanamycin (final concentration 3 (g / ml)), positive clones were screened by colony PCR method and sequenced. Correct positive clone (pET-0595c0 2 ) The plasmid was transformed into Escherichia coli BL21 (DE3) P lySs (Nova g en).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex.
  • hemocyanin and bovine serum albumin For methods, see: Avrameas, et al. Ira wake ochemistry, 1969; 6: 43. Rabbits were immunized with 4 mg of the hemocyanin-polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin-polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once.
  • a titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum.
  • Total IgG was isolated from antibody-positive rabbit sera using protein A-Sepharose.
  • the peptide was bound to a cyanogen bromide-activated Sepharos B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography.
  • the immunoprecipitation method demonstrated that the purified antibody specifically binds to ribosomal protein L22 and sperm activating peptide receptor repeat domain 12.
  • the suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in various aspects.
  • the probes can be used to hybridize to the genome or CDM library 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 tissues or 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 using a filter hybridization method.
  • Filter membrane hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods, etc., all of which fix the polynucleotide sample to be tested on the filter The membranes were hybridized using essentially the same procedure.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer, so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthetic polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • the GC content is 30% -70%, and the non-specific hybridization increases when it exceeds;
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, then the primary probe should not be used;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 1 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 membranes nitrocellulose membranes
  • Two NC membranes are required for each probe, so that they can be used in the following experimental steps.
  • the film was washed with high-strength conditions and strength conditions, respectively.
  • the sample membrane was placed in a plastic bag, and 3-10 mg of prehybridization solution (10xDenhardt> s; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA)) was added. After closing the bag, 68. C water bath for 2 hours.
  • High-intensity washing film 1) Take out the hybridized sample membrane.

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Abstract

L'invention concerne de nouveaux polypeptides, un domaine de répétition 12 de récepteurs peptidiques de la motilité du sperme et une protéine ribosomale L22, et des polynucléotides codant ces polypeptides ainsi qu'un procédé d'obtention de ces polypeptides par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ces polypeptides dans le traitement de maladies, notamment d'athétoses, de tumeurs malignes, de l'hémopathie, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi les antagonistes agissant contre les polypeptides et leur action thérapeutique ainsi que les applications de ces polynucléotides codant le domaine de répétition 12 de récepteurs peptidiques de la motilité du sperme et la protéine ribosomale L22.
PCT/CN2001/000915 2000-06-07 2001-06-04 Nouveaux polypeptides, domaine de repetition 12 de recepteurs peptidiques de la motilite du sperme et proteine ribosomale l22, et polynucleotides codant ces polypeptides WO2002002611A1 (fr)

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CN 00116372 CN1326983A (zh) 2000-06-07 2000-06-07 一种新的多肽——核糖体蛋白l22及精子活化肽受体重复域12和编码这种多肽的多核苷酸
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1637120A2 (fr) 2004-09-16 2006-03-22 Kimberly-Clark Worldwide, Inc. Formulations de poudres liquides, aqueuses et non alcooliques
WO2006095355A1 (fr) * 2005-03-07 2006-09-14 Ganga Raju Gokaraju Nouveaux sels d’acides boswelliques, acides boswelliques selectivement enrichis et procede de preparation de ceux-ci

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BIOCHIM. BIOPHYS. ACTA, vol. 1260, no. 1, 1995, pages 113 - 115 *
GENE, vol. 154, no. 2, 1995, pages 199 - 203 *
J. BIOL. CHEM., vol. 259, no. 22, 1984, pages 13712 - 13716 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1637120A2 (fr) 2004-09-16 2006-03-22 Kimberly-Clark Worldwide, Inc. Formulations de poudres liquides, aqueuses et non alcooliques
WO2006095355A1 (fr) * 2005-03-07 2006-09-14 Ganga Raju Gokaraju Nouveaux sels d’acides boswelliques, acides boswelliques selectivement enrichis et procede de preparation de ceux-ci

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