WO2001081381A1 - Nouveau polypeptide, proteine humaine 9 contenant un fragment de sequence particulier d'une recombinase specifique au site, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, proteine humaine 9 contenant un fragment de sequence particulier d'une recombinase specifique au site, et polynucleotide codant pour ce polypeptide Download PDF

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WO2001081381A1
WO2001081381A1 PCT/CN2001/000579 CN0100579W WO0181381A1 WO 2001081381 A1 WO2001081381 A1 WO 2001081381A1 CN 0100579 W CN0100579 W CN 0100579W WO 0181381 A1 WO0181381 A1 WO 0181381A1
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polypeptide
sequence
protein
polynucleotide
human
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PCT/CN2001/000579
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English (en)
Chinese (zh)
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Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
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Priority to AU73790/01A priority Critical patent/AU7379001A/en
Publication of WO2001081381A1 publication Critical patent/WO2001081381A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, a human-containing protein 11 containing a site-specific recombinase characteristic sequence fragment, and a polynucleotide sequence encoding the polypeptide. The invention also relates to methods and applications for preparing such polynucleotides and polypeptides. Background technique
  • Site-specific recombinases play an important regulatory role in the process of prokaryotic DNA recombination.
  • DNA recombination is an important step in the reorganization and evolution of genetic material. People have cloned two different types of recombinases: one plays a regulatory role in reversing the recombination of DNA fragments; the other plays a regulatory role in the excision and recombination of DM fragments.
  • DNA site-specific recombination requires only single-stranded transformations, without the need for DNA synthesis or high energy co-factors.
  • members of the lysozyme family consist of 180 to 200 amino acid residues.
  • protein sequence of the protein family members contains two highly conserved characteristic sequence fragments.
  • the first conserved sequence fragment is the N-terminal region of the protein, which contains a conserved serine residue, which is involved in the covalent relationship between protein and DNA. Binding; a second conserved characteristic sequence fragment is approximately 50 amino acid residues upstream of the serine active site.
  • Sequence fragment 1 Y- [LIVAC] -R- [VA] -S- [ST] -X (2)-Q; Sequence fragment 2: G- [DE ] -X (2)-[LIVM] -X (3)-[LIVM]-[DT] -R- [LIVM]-[GSA]; where S in sequence fragment 1 is the serine active center site.
  • the mutation or abnormal expression of the two sequence fragments will directly affect the function of the direct protein in the body, and then affect the recombination and expression of related DNA genetic material in the body.
  • DM recombinase can be divided into two different protein families in the body.
  • the members of the lysozyme family are widely distributed in the body, and they are involved in regulating a variety of important DM recombination and evolution processes. Mutations or abnormal expression will directly affect the recombination and expression of genetic material in the body.
  • Members of this protein family are usually associated with a variety of developmental disorders, hereditary diseases and related tissue tumors and cancers in the organism. The occurrence of disease is closely related.
  • the human protein 11 protein containing the site-specific recombinase characteristic sequence fragment plays an important role in important functions in the body, and it is believed that a large number of proteins are involved in these regulatory processes, so more needs to be identified in the art Those involved in these processes are protein 11 proteins containing site-specific recombinase-specific sequence fragments, particularly the amino acid sequence of this protein.
  • the newcomer's protein containing site-specific recombinase-specific sequence fragments 11 The isolation of the protein-coding gene also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for the disease, so it is important to isolate its coded DM. Disclosure of invention
  • An object of the present invention is to provide an isolated novel polypeptide-to-human protein 11 containing site-specific recombinase characteristic sequence fragments, and fragments, analogs and derivatives thereof.
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a protein 11 containing a human-containing site-specific recombinase-specific sequence fragment.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a protein 11 containing a human-containing site-specific recombinase characteristic sequence fragment.
  • Another object of the present invention is to provide a method for producing human protein 11 containing a site-specific recombinase-specific sequence fragment.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention, a human-containing protein 11 containing a site-specific recombinase-specific sequence fragment.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors of the protein 11 of the polypeptide of the present invention, which contain a site-specific recombinase-specific sequence fragment protein 11.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities in protein 11 of human-containing site-specific recombinase characteristic sequence fragments.
  • 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 435-746 in SEQ ID NO: 1; and (b) a sequence having 1-1597 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 present invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of human protein 11 protein containing site-specific recombinase-specific sequence fragments, which comprises using the polypeptide of the present invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for detecting a disease or disease susceptibility related to abnormal expression of a protein 11 protein containing a site-specific recombinase characteristic sequence fragment in human in vitro, which comprises detecting the polypeptide or a polynucleoside encoded therein in a biological sample. Mutations in the acid sequence, 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 abnormal expression of the protein 1 1 of the polypeptide and / or polynucleotide of the present invention in the preparation of protein 1 1 for treating cancer, developmental disease or immune disease or other humans containing site-specific recombinase characteristic sequence fragments. Use of drugs that cause disease.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genome or a 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 a fragment or part 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 may have "conservative" changes in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine.
  • Variants are also available Has non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind to specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that causes a change in the protein and thereby regulates the activity of the protein when it binds to a protein 11 containing a site-specific recombinase characteristic sequence fragment.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other protein that binds to a fragment containing human site-specific recombinase-specific sequences
  • Antagonist refers to a type of human-site-specific recombinase-specific recombinase-specific sequence fragment that binds or regulates human-site-specific recombinase-specific sequence fragments when combined with protein 11.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that binds to human 11 containing a site-specific recombinase characteristic sequence fragment.
  • Regular refers to a change in the function of human 11 containing a site-specific recombinase characteristic sequence fragment, including an increase or decrease in protein activity, a change in binding characteristics, and a human site-specific recombinase characteristic sequence. Alteration of any other biological, functional or immune properties of the fragment of protein 11.
  • Substantially pure means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can use standard protein purification techniques to purify human proteins containing site-specific recombinase-specific sequence fragments11.
  • Substantially pure human proteins containing site-specific recombinase-specific sequence fragments 11 produce a single main band on a non-reducing polyacrylamide gel.
  • the purity of the human 11-site-specific recombinase-specific sequence fragment protein 11 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of a nucleotide by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence "C-T-G-A” can be combined with the complementary sequence "G-A-C-T”.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. Inhibition of this hybridization can be achieved by hybridization under conditions of reduced stringency (Southern or Northern blotting). Etc.) to detect.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are the same or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Mad Son Wis.). The MEGALIGN program can compare two or more sequences according to different methods, such as Cluster's 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 the following formula: The number of matching residues between sequence A and sequence X 100
  • nucleic acid sequences can also be determined by the Clus ter method or by a method known in the art such as Jotun He in (He in J., (1990) Methods in emzumo l ogy 183: 625-645).
  • Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitutions for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to the “sense strand”.
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the primary biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa, F (ab ') 2 and Fv, which can specifically bind to the human epitope of protein 11 containing site-specific recombinase characteristic sequence fragments.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it occurs naturally).
  • a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not a component of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances in the natural state .
  • isolated human site-specific recombinase characteristic sequence fragment protein 11 refers to human site-specific recombinase characteristic sequence fragment protein 11 that is substantially free of other proteins naturally associated with it , Lipids, sugars or other substances.
  • Those skilled in the art can use standard protein purification techniques to purify human proteins containing site-specific recombinase-specific sequence fragments 11.
  • Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel.
  • the purity of the human 11-site-specific recombinase characteristic sequence fragment protein 11 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a novel polypeptide-human protein 11 containing a site-specific recombinase characteristic sequence fragment, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeasts, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated, or may be non-glycosylated. Polypeptides of the invention may also include or exclude initial methionine residues.
  • the present invention also includes fragments, derivatives, and analogs of human protein 11 containing a fragment specific for a recombinase characteristic sequence.
  • fragment refers to a protein 11 that substantially maintains the same biological function or activity of a human-containing site-specific recombinase characteristic sequence fragment of the present invention. Peptide.
  • 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 ( ⁇ ) 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 the mature polypeptide is fused with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV) a type in which the additional amino acid sequence is fused into the mature polypeptide and shaped Into a polypeptide sequence (such as a leader sequence or a secreted sequence or a sequence used to purify the polypeptide or a protein sequence). As described herein, 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 cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 1597 bases in length and its open reading frame (435-746) encodes 103 amino acids.
  • This peptide has the characteristic sequence of the DNA recombinase protein family, and it can be deduced that the human protein 11 containing the site-specific recombinase characteristic sequence fragment has the structure and function represented by the MA recombinase protein family.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DM.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide that encodes the polypeptide and a polynucleotide that includes additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity between the two sequences).
  • the invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions.
  • “strict conditions” means: (1) 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% F ico ll, 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, 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 protein 11 of humans containing site-specific recombinase-specific sequence fragments.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence of the protein 11 encoding a site-specific recombinase-specific sequence fragment of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DM 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 mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • Various methods have been used to extract mRNA, and kits are also commercially available (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 combined with polymerase reaction technology, even very small expression products can be cloned.
  • the genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (1) DNA-DM or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of human proteins containing site-specific recombinase-specific sequence fragments The level of the transcript of 11; (4) detecting the protein product of the gene expression by immunological techniques or measuring biological activity. The above methods can be used alone 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 DM sequence chemically synthesized based on the gene sequence information of the present invention. The genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product that detects the expression of the protein 11 gene of the human-containing site-specific recombinase characteristic sequence fragment can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) and so on.
  • a method 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. Selected and synthesized by conventional methods.
  • the amplified DNA / RM fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, the sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a genetically engineered host cell using the vector of the present invention or directly using a protein 11 coding sequence containing a site-specific recombinase characteristic sequence fragment of human, and recombinant Technology A method of producing a polypeptide of the invention.
  • a polynucleotide sequence encoding a protein 11 containing a human-specific site-specific recombinase characteristic sequence fragment can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain replication origins, promoters, marker genes, and translational regulatory elements.
  • Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequence of protein 11 encoding human site-specific recombinase-specific sequence fragments and suitable transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, retroviral LTRs and other known controllable genes in prokaryotes A promoter expressed in a cell or eukaryotic cell or its virus.
  • 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 SV40 enhancer of 100 to 2 70 base pairs on the late side of the origin of replication, polyoma enhancer and adenovirus enhancer on the late side of the origin of replication, and the like.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human protein 11 containing a site-specific recombinase characteristic sequence fragment or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute the polynucleotide or the recombinant.
  • 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 fly S2 or Sf 9
  • animal cells such as CHO, COS or Bowes s melanoma cells, etc. .
  • Transformation of a host cell with a DM sequence according to the present invention or a recombinant vector containing the DNA sequence can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of DNA uptake can be in the exponential growth phase were harvested, treated with CaC l 2 method used in steps well known in the art. The alternative is to use MgC l 2 .
  • transformation can also be performed by electroporation.
  • the 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 human site-specific recombinase-specific sequence fragment protein 1 1 (Sc ience, 19 84; 224: 14 31 ). Generally there are the following steps:
  • a polynucleotide (or variant) of the present invention encoding a protein 1 1 fragment containing a human-specific site-specific recombinase characteristic sequence fragment, or transformed or transformed with a recombinant expression vector containing the polynucleotide Direct suitable host cells; (2) culturing host cells in a suitable medium;
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromat
  • FIG. 1 is a comparison diagram of the amino acid sequence homology of the protein 11 of the present invention containing a site-specific recombinase characteristic sequence fragment at a total of 45 amino acids from 8 to 52 and a characteristic domain of the DNA recombinase protein family.
  • the upper sequence is human protein 11 containing site-specific recombinase-specific sequence fragments, and the lower sequence is the characteristic domain of the DNA recombinase protein family.
  • Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+".
  • Figure 2 is a polyacrylamide gel electrophoresis image (SDS-PAGE) of an isolated human protein 11 containing a site-specific recombinase characteristic sequence fragment.
  • llKDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Use Smart cDNA Cloning Kit purchased from Clontech. The 0 ⁇ fragment was inserted into the multicloning site of the pBSK (+) vector (Clontech), and transformed into DH5 ⁇ to form a CDM library.
  • Dye terminate cycle reaction 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 1283a08 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 protein 11 containing the site-specific recombinase characteristic sequence fragment of the present invention and its encoded protein sequence ⁇ 1 J were used with the profile scan program (Bas iclocai Alignment search tool) in GCG [Altschul, SF et al. J. Mol. Biol. 1990; 215: 403-10], domain analysis was performed in databases such as prosite.
  • the human 11 containing the site-specific recombinase characteristic sequence fragment protein 11 at 8-52 is homologous with the domain DNA recombinase protein family. The results of the homology are shown in Fig. 1. The homology is 0.17, and the score is 7.72; The threshold is 7.61.
  • Example 3 Cloning of a gene encoding human 11 containing a site-specific recombinase characteristic sequence fragment by RT-PCR
  • CDM 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, PCR was performed using the following primers for PCR amplification:
  • Primer 1 5'-GGGCCGCCAAGGCGGGGGAGGCCG-3 '(SEQ ID NO: 3)
  • Primer2 5'-CATAGGCCGAGGCGGCCGACATGT-3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp of the 5th end of SEQ ID NO: 1;
  • Primer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.
  • Amplification conditions 50 mmol / L KC1, 10 mmol / L in a 50 ⁇ l reaction volume
  • Tris-Cl (pH 8.5), 1.5 mmol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol primer, 1U Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 55. C 30sec; 72 ° C 2rain. Simultaneously set ⁇ during RT-PCR -act in is the positive control and template blank is the negative control.
  • the amplified product was purified using a QIAGEN kit and ligated to a pCR vector using a TA cloning kit (Invitrogen).
  • RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] 0
  • This method involves acid guanidinium thiocyanate-chloroform extraction. I.e. with 4M guanidine isothiocyanate - 25mM sodium citrate, 0.2M sodium acetate (P H4.0) of the tissue was homogenized, 1 volume of phenol and 1/5 volume of chloroform - isoamyl alcohol (49: 1) After mixing, centrifuge. 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.
  • a 32P-labeled probe (about 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 / nil salmon sperm DNA. After hybridization, the filters were placed 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 Human Site-Specific Recombinase Characteristic Sequence Fragment Protein 11
  • Primer 3 5-CCCCATATGATGAAAATGAAAGAAGAAAAGAGA-3 '(Seq ID No: 5)
  • Primer4 5-CATGGATCCCTACTGGACTGGACACATCCACAT-3 '(Seq ID No: 6)
  • the 5' ends of these two primers contain Ndel and BamHI digestion sites, respectively, followed by the coding sequences of the 5 'and 3' ends of the target gene, respectively
  • BamHI restriction sites correspond to selective endonuclease sites on the expression vector plasmid pET 28b (+) (Novagen, Cat. No. 69865.3).
  • a PCR reaction was performed using the pBS-1283a08 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ containing pBS-1283a08 plasmid 10 pg, primers Primer-3 and Primer-4 were lOpmol 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. Do not recover large fragments and ligate with T4 ligase. The ligation product was transformed into E. coli DH5a by the calcium chloride method.
  • the bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. The affinity chromatography column His.Bind Quick Cartridge (product of Novagen) was used to obtain 6 histidines (6His-Tag).
  • the purified target protein 11 containing the site-specific recombinase-specific sequence fragment was purified. After SDS-PAGE electrophoresis, a single band was obtained at 11 kDa ( Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by the Edams hydrolysis method.
  • NH2-Met-Lys-Met-Lys-Glu-Glu-Lys-Arg-Asp-Thr-Phe-Asn-Val-Ser-Ser-C00 H (SEQ ID NO: 7).
  • 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. Immunochemistry, 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 the immunity once.
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this example is to select a suitable oligonucleoside from the polynucleotide SEQ ID NO: 1 of the present invention
  • the acid fragment is used as a hybridization probe, and the membrane hybridization method is used to identify whether some tissues contain the polynucleotide sequence of the present invention or a homologous polynucleotide sequence.
  • Filter hybridization methods include dot blotting, Southern blotting, Nor thern blotting, and copying methods. They are all used to fix the polynucleotide sample to be tested on the filter and then hybridize using basically the same steps.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment 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 (pr 0 bel), which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 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 7 Resuspend the DNA pellet in a small volume of TE or water. Vortex at low speed or suck with a dropper, and gradually increase TE, mix until the DNA is fully dissolved, and add approximately lul for each 1-5 x 10 phasic cells.
  • steps 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 in order to follow the experimental steps.
  • the film was washed with high-strength conditions and strength conditions, respectively.
  • the 32 P-Probe (the second peak is free ⁇ - 32 P-dATP) is prepared after combining the collection solutions of the first peak.
  • prehybridization solution 10xDenhardt's; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA).
  • polypeptides of the present invention as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.
  • Site-specific recombinases play important regulatory roles in biological DNA recombination processes. Such as the cycle-integrated recombinase family, the lyase family. Recombinase family members are involved in many important DNA recombination processes in the body. Mutations or abnormal expression of the conserved sequence fragments of the members of the recombinase family will directly affect the function of the direct protein in vivo, and then affect the reorganization and expression of related DNA genetic material in vivo.
  • the polypeptide of the present invention is a polypeptide containing a characteristic sequence of the recombinant enzyme protein family, and has the basic biological function of the protein of the recombinant enzyme family. Abnormal expression of the polypeptide will lead to abnormal regulation of growth and development of the body, and also involves abnormal regulation of cell division and proliferation. Cause related diseases.
  • the abnormality will produce various diseases, especially growth disorders, various tumors, embryonic development disorders, inflammation, and immune diseases. These diseases include, but are not limited to:
  • Growth and development disorders mental retardation, brain development disorders, skin, fat, and muscular dysplasia, bone and joint dysplasia, various metabolic defects, stunting, dwarfism, Cushing's syndrome Sexual retardation
  • 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, colon cancer, thymic tumor, nasopharyngeal cancer, laryngeal cancer, tracheal tumor, fibroid, fibrosarcoma, lipoma, liposarcoma
  • Embryonic disorders congenital abortion, cleft palate, limb loss, limb differentiation disorder, atrial septal defect, neural tube defect, congenital hydrocephalus, congenital glaucoma or cataract, congenital deafness
  • 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
  • the abnormal expression of the protein 11 containing the site-specific recombinase characteristic sequence fragment of the present invention will also cause certain hereditary, hematological diseases and the like.
  • polypeptide of the present invention can be directly used in the treatment of diseases, for example, it can treat various diseases, especially growth and development disorders, various tumors, embryonic development disorders, inflammation, and immunity. Sexual diseases, certain hereditary, blood diseases, etc.
  • the present invention also provides methods for screening compounds to identify agents that enhance (agonist) or suppress (antagonist) human proteins containing site-specific recombinase-specific sequence fragments 11.
  • Agonists enhance human proteins containing site-specific recombinase-specific sequence fragments to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • a membrane preparation of a mammalian cell or a protein 11 expressing a human site-specific recombinase-specific sequence fragment and a labeled human site-specific recombinase-specific sequence fragment protein can be used. 11 Cultivate together. The ability of the drug to increase or suppress this interaction is then determined.
  • Antagonists of human protein 11 containing site-specific recombinase characteristic sequence fragments include screened antibodies, compounds, receptor deletions, and the like.
  • Antagonist of human site-specific recombinase characteristic sequence fragment protein 11 antagonist can bind to human site-specific recombinase characteristic sequence fragment protein 11 and bind Eliminating its function, or inhibiting the production of the polypeptide, or binding to the active site of the polypeptide prevents the polypeptide from performing its biological function.
  • human proteins containing site-specific recombinase-specific sequence fragments 11 can be added to the bioanalytical assay, and the compounds can be used to determine the site-specific recombinase-characteristic sequence fragments of humans.
  • Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to human protein 11 containing site-specific recombinase characteristic sequence fragments can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. In screening, generally, human 11 molecules containing site-specific recombinase characteristic sequence fragments should be labeled.
  • the present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the present invention also provides antibodies directed against a protein 11 epitope containing a site-specific recombinase characteristic sequence fragment of human. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human proteins containing a site-specific recombinase characteristic sequence fragment protein 11 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.
  • Techniques for the preparation of human monoclonal antibodies containing protein 11 fragment specific for the recombinase characteristic sequence include, but are not limited to, hybridoma technology (Kohler and Mil te in. Nature, 1975, 256: 495-497) , Three tumor technology, human B-cell hybridoma technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions and non-human-derived variable regions can be produced using existing techniques (Morrison et al., PNAS, 1985, 81: 6851).
  • the existing technology for producing single-chain antibodies U.S. Pat No. 4946778, can also be used to produce single-chain antibodies against human 11 containing a site-specific recombinase characteristic sequence fragment.
  • Antibodies against protein 11 containing site-specific recombinase-specific sequence fragments of humans can be used in immunohistochemistry to detect human site-specific recombinase-specific sequence fragments of protein 11 in biopsy specimens.
  • Monoclonal antibodies that bind to protein 11 containing human-specific site-specific recombinase-specific sequence fragments can also be labeled with radioisotopes and injected into the body to track their location and distribution.
  • This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • a human 11 high-affinity monoclonal antibody containing a site-specific recombinase characteristic sequence fragment can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to use a thiol crosslinking agent such as SPDP, which attacks the amino group of an antibody, binds the toxin to the antibody through the exchange of disulfide bonds.
  • SPDP thiol crosslinking agent
  • This hybrid antibody can be used to kill human protein 11-positive cells containing site-specific recombinase-specific sequence fragments.
  • the antibodies of the present invention can be used to treat or prevent diseases related to protein 11 containing human-specific site-specific recombinase characteristic sequence fragments.
  • Administration of appropriate doses of antibodies can stimulate or block the production or activity of human protein 11 containing site-specific recombinase characteristic sequence fragments.
  • the present invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human protein 11 containing a site-specific recombinase characteristic sequence fragment.
  • tests are well known in the art and include FI SH assays and radioimmunoassays.
  • the level of protein 11 of human site-specific recombinase characteristic sequence fragments detected in the test can be used to explain the importance of human site-specific recombinase characteristic sequence fragments of protein 11 in various diseases and It is used to diagnose a disease in which human protein 11 containing a site-specific recombinase characteristic sequence fragment functions.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry.
  • Polynucleotides encoding a protein 11 containing human site-specific recombinase characteristic sequence fragments can also be used for a variety of therapeutic purposes.
  • Gene therapy technology can be used to treat abnormal cell proliferation, development, or metabolism caused by the non-expression or abnormal / inactive expression of protein 11 containing human-specific site-specific recombinase-specific sequence fragments.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human site-specific recombinase-specific sequence fragments of protein 11 to inhibit endogenous human site-specific recombinase-specific sequences Fragment of protein 11 activity.
  • a mutated human site-specific recombinase characteristic sequence fragment protein 11 may be a shortened, human-site-specific recombinase characteristic sequence fragment protein 11 lacking a signaling domain, although Can bind to downstream substrates, but lacks signaling activity. Therefore, recombinant gene therapy vectors can be used to treat human proteins containing site-specific recombinase-specific sequence fragments.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding a protein 11 encoding a human-containing site-specific recombinase-specific sequence fragment Into the cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a protein 11 containing a site-specific recombinase characteristic sequence fragment can be found in the existing literature (Sambrook, et al.).
  • a polynucleotide encoding human protein 11 containing a site-specific recombinase-specific sequence fragment 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 that inhibit human protein 11 mRNA containing site-specific recombinase-specific sequence fragments are also within the scope of the invention.
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA to perform endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as the technology for the synthesis of oligonucleotides by solid-phase phosphoramidite chemical synthesis has been widely used.
  • Antisense RM molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RM. This DM sequence has been integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of a nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside linkages should use phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • Polynucleotides encoding human site-specific recombinase-specific sequence fragment protein 11 can be used for the diagnosis of diseases related to human site-specific recombinase-specific sequence fragment protein 11.
  • Polynucleotides encoding protein 11 of human site-specific recombinase characteristic sequence fragments can be used to detect the expression of human site-specific recombinase characteristic sequence fragments of protein 11 or human-containing sites in disease states Dot-Specific Recombinase Characteristic Sequence Fragment Abnormal Expression of Protein 11.
  • a DNA sequence encoding a protein 11 containing human site-specific recombinase characteristic sequence fragments can be used to hybridize biopsy specimens to determine the expression status of human site-specific recombinase characteristic sequence fragments of protein 11.
  • 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 polynucleotide of the present invention can be fixed as a probe on a micro array or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis of genes and genetic diagnosis in tissues.
  • Human site-specific recombinase characteristic sequence fragment protein 11 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect human site-specific recombinase characteristic sequence fragment protein 11 transcript.
  • Detecting mutations in the protein 11 gene containing human site-specific recombinase-specific sequence fragments can also be used to diagnose human protein 11-containing disease-specific recombinase-characterizing sequence fragments.
  • Human 11 site-specific recombinase characteristic sequence fragment protein 11 mutation forms include point mutations, translocations, and deletions compared to normal wild-type human site-specific recombinase characteristic sequence fragment protein 11 DM sequences , Reorganization, and any other abnormalities. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, the Nor thern imprinting method and Western blotting method can be used to indirectly determine whether a gene is mutated.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for labeling chromosome positions.
  • it The important first step is to locate these DM sequences on the 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 DNA to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendel ian Inheritance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the 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 diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients that do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • the polypeptides of the present invention can be combined with other therapeutic agents. Conjugates are used in combination.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human protein containing a fragment specific for a recombinase characteristic sequence 11 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dosage range of human 11 containing the site-specific recombinase-specific sequence fragment protein 11 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine humaine 9 contenant un fragment de séquence particulier d'une recombinase spécifique au site, et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, des troubles du développement, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la protéine humain 9 contenant un fragment de séquence particulier d'une recombinase spécifique au site.
PCT/CN2001/000579 2000-04-27 2001-04-23 Nouveau polypeptide, proteine humaine 9 contenant un fragment de sequence particulier d'une recombinase specifique au site, et polynucleotide codant pour ce polypeptide WO2001081381A1 (fr)

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CN 00115494 CN1320697A (zh) 2000-04-27 2000-04-27 一种新的多肽——人含位点特异的重组酶特征性序列片段的蛋白11和编码这种多肽的多核苷酸

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Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BRECHT S. ET AL., GENE, vol. 234, no. 2, 8 July 1999 (1999-07-08), pages 239 - 247 *
CHEN L. ET AL., SOMAT. CELL. MOL. GENET., vol. 22, no. 6, November 1996 (1996-11-01), pages 477 - 488 *
FLOWERS C.C. ET AL., J. VIROL., vol. 71, no. 4, April 1997 (1997-04-01), pages 2685 - 2692 *
NICHOLS M. ET AL., MOL. ENDOCRINOL., vol. 11, no. 7, June 1997 (1997-06-01), pages 950 - 961 *
ROSSI F. ET AL., CURR. BIOL., vol. 6, no. 7, 1 July 1996 (1996-07-01), pages 866 - 872 *

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