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WO2001030835A1 - Nouveau polypeptide, pogo transposase humaine 14, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, pogo transposase humaine 14, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001030835A1
WO2001030835A1 PCT/CN2000/000353 CN0000353W WO0130835A1 WO 2001030835 A1 WO2001030835 A1 WO 2001030835A1 CN 0000353 W CN0000353 W CN 0000353W WO 0130835 A1 WO0130835 A1 WO 0130835A1
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Prior art keywords
polypeptide
transposase
human
polynucleotide
pogo
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Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Bioroad Gene Development Ltd
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Shanghai Bioroad Gene Development Ltd
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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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, human pogo transposase 14, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a method and application for preparing the polynucleotide and polypeptide.
  • Transposable elements also known as transposons, are DM sequences that can be transferred between different chromosomes in the same cell, or between different sites on the same chromosome. This transfer does not depend on the homology between the sequences.
  • transposons contain an open reading frame (0RF), which may encode a transposase.
  • ITRs terminal inverted repeats
  • a short sequence of the target sequence on the recipient DNA Due to the insertion of the transposon, the target sequence forms a forward repeat on both sides of the transposon.
  • the length of the target sequence is specific to each type of transposon.
  • transposase In translocation, a transposase is required.
  • the transposase is encoded by the open reading frame of the transposon, and may also be encoded by other genes, and its role is to catalyze the transposition of the transposon.
  • transposons can be divided into many families.
  • One is the Mariner superfamily.
  • the Mariner transposon moved on the DNA intermediate using a "cut and paste" mechanism.
  • the transposon was cut from the original place of the DNA and inserted into the new position of the genome.
  • the insertion target of the Mariner transposon is a TA dinucleotide sequence, and this dinucleotide sequence is copied when the Mariner transposon is inserted.
  • the domain unique to this family of transposases is the "D, D35E” structure, which includes a conserved Asp and a “D35E” region.
  • the "D35E” region contains conserved arginine and glutamate residues, and there are generally 35 relatively conserved amino acid residues in between.
  • the Mariner transposon family is thought to be associated with many diseases.
  • the ⁇ .2 site was found to be replicated in progressive neurofibular muscle atrophy 1A, and in hereditary neuropathic paralysis, this site has 1.
  • structures similar to Mariner transposons can be found near this site [Hart l (1996)]. This structure-encoded transposase may participate in the unbalanced exchange of 17 ⁇ 11 ⁇ 2, resulting in the replication or deletion of this site.
  • the human gene of the present invention has 77% homology at the protein level with the transposase encoded by the fruit fly pogo transposon ORF1. It is very similar to the D, D35E domain, a characteristic domain of the Mariner transposase family. Based on the above points, the new gene of the present invention is considered to be a gene encoding human and Drosophila pogo transposon subfamily D, D35E domains, and named human pogo transposase 14. It is inferred that it is similar to D and D35E domains and has similar biological functions.
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding human pogo transposase 14.
  • Another object of the present invention is to provide a method for producing human Pogo transposase 14.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention, human pogo transposase 14.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the polypeptide of the present invention, human Pogo transposase 14.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of human pogotransposase 14.
  • a novel isolated human pogo transposase 14 is provided.
  • the polypeptide is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID NO: 2, or a conservative variant polypeptide thereof, or Its active fragment, or its active derivative, analog.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2 .
  • a polynucleotide encoding the isolated polypeptides, the polynucleotide comprising a nucleotide sequence having at least 78 nucleotides with a nucleotide sequence selected from the group consisting of % Identity: (a) a polynucleotide encoding the aforementioned human pogo transposase 14; (b) a polynucleotide complementary to the polynucleotide (a).
  • the polynucleotide encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 2.
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 538-930 in SEQ ID NO: 1; and (b) having a sequence of 1-2421 in SEQ ID NO: 1 Sequence of bits.
  • FIG. 1 is a comparison diagram of amino acid sequence homology between the inventor's pogo transposase 14 and the Drosophila transposase.
  • the upper sequence is human pogo transposase 14, and the lower sequence is Drosophila transposase.
  • 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 the isolated human pogo transposase 14.
  • 14kDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • 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 pogo transposase 14 means that human pogo transposase 14 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify human pogo transposase 14 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the human pogo transposase 14 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human pogo transposase 14, which basically consists of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be obtained from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammals) using recombinant technology. Biological cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of human pogo transposase 14.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the human pogo transposase 14 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or ( ⁇ )
  • Such a polypeptide sequence 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
  • a polypeptide sequence in which an 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 the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue.
  • the full-length polynucleotide sequence contains 2421 bases, and its open reading frame (538-930) encodes 1 30 amino acids. According to the amino acid sequence homology comparison, it was found that this polypeptide has 77% homology with Drosophila transposase. It can be inferred that the human pogo transposase 14 has a similar structure and function to Drosophila transposase.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be days Naturally occurring allelic 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 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, 6 (TC; or (2) added during hybridization Use a denaturing agent, such as 50 ° /. (V / v) formamide, 0.1% calf serum / 0.1 ° /.
  • 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 cores. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques such as PCR to identify and / or isolate polynucleotides encoding human pogo transposase 14.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding the human pogo transposase 14 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 D 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.
  • mRNA extraction There are many mature techniques for mRNA extraction, and kits are also commercially available (Qiagene).
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes can be screened from these cDNA libraries by conventional methods. These methods include (but not Limited to): (l) DNA-DM or DNA-RNA hybridization; (2) the presence or absence of a marker gene function; (3) determination of the level of human pogo transposase 14 transcripts; (4) through immunological techniques or The biological activity is measured to detect the protein product expressed by the gene.
  • 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 1 000 nucleotides.
  • the probe used herein is usually a D sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELI SA) can be used to detect the protein product expressed by the human pogo transposase 14 gene.
  • ELI SA enzyme-linked immunosorbent assay
  • a method for amplifying DNA / RM using PCR technology (Saiki, etal. Sc; 1985; 230: 1 350-1 354) is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers 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 D fragments and the like obtained as described above can be measured by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a human pogo transposase 14 coding sequence, and a method for producing the polypeptide of the present invention by recombinant technology. .
  • a polynucleotide sequence encoding human pogo transposase 14 may be inserted into a vector to form a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors expressed in bacteria (Rosenberg, et al.
  • pMSXND expression vectors expressed in mammalian cells Lee and Na thans, J Bio Chem. 263: 3521, 1988
  • baculovirus-derived vectors expressed in insect cells in short, as long as it can be replicated and stabilized in a host, any plasmid and vector can be used to construct a recombinant expression vector.
  • An important expression vector It is characterized by containing replication origins, promoters, marker genes and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human pogo transposase 14 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology (Sambroook, etal. Mo lecu l ar C l on ing, a Labora t ory Manua l, co ld Spr ing Harbor Labora tory. 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: l ac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation, a transcription terminator, and the like. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Examples include SV40 enhancers of 100 to 270 base pairs 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 human pogo transposase 14 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf 9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • 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 harvested after exponential growth phase, with (: Treatment 1 2, steps well known in the art used alternative is to use MgCl 2..
  • transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DM transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposomes Packaging, etc.
  • polynucleotide sequence of the present invention can be used to express or produce recombinant human pogo transposase 14 (Science, 1984; 224: 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted 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 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.
  • Mariner transposon-like structures have been found in diseases such as progressive neurofibular muscular atrophy 1A and hereditary neuropathic paralysis.
  • the transposase encoded by this structure is thought to play a role in the unbalanced replication or deletion of 17 pl 1.2. Therefore, peptides homologous to Mariner transposase can be used to treat and prevent diseases such as progressive neurofibular muscular atrophy 1A and hereditary neuropathic paralysis, as well as other related diseases.
  • the present invention can also be used to treat or prevent the loss or inferiority of gene function caused by gene deletion or duplication caused by transposase transposition.
  • Antagonists or a fragment or derivative of human pogo transposase 14 can be used to treat or prevent cancer, including but not limited to adenocarcinoma, leukemia, lymphoma, melanoma, sarcoma, myeloma, teratoma, Adrenal cancer, bladder cancer, bone cancer, bone marrow cancer, brain cancer, breast cancer, uterine cancer, gallbladder cancer, nerve center cancer, kidney cancer, liver cancer, lung cancer, thyroid cancer, thymic cancer, etc.
  • Antibodies that specifically bind to human pogo transposase 14 can be used directly as antagonists or indirectly to bring the agent to cells or tissues expressing human pogo transposase 14 in a targeting or delivery mechanism.
  • Antagonist or a fragment of human pogo transposase 14 Or derivatives can be used to treat or prevent cancer immune disorders.
  • the translocation effect of the present invention can be used to introduce normal genes into target cells, stably express normal proteins and make the disease alleviate or cure.
  • the invention also provides screening compounds to identify improvements
  • Agonist or method of repressing (antagonist) human pogo transposase 14 agent.
  • Agonists enhance human pogo transposase 14 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 a membrane preparation expressing human pogo transposase 14 can be cultured with labeled human pogo transposase 14 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human pogo transposase 14 include antibodies, compounds, receptor deletions, and analogs that have been screened. Antagonists of human pogo transposase 14 can bind to human pogo transposase 14 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions.
  • human pogo transposase 14 When screening compounds as antagonists, human pogo transposase 14 can be added to a bioanalytical assay to determine whether the compound is an antagonist by measuring the effect of the compound on the interaction between human pogo transposase 14 and its receptor. . Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds. Polypeptide molecules capable of binding to human pogo transposase 14 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. In screening, human pogo transposase 14 molecules should generally 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 human pogo transposase 14 epitopes. These antibodies include (but are not limited to): Doklon antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, and fragments from Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human pogo transposase 14 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant. Wait.
  • Techniques for preparing monoclonal antibodies to human pogo transposase 14 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridization Tumor technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions 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 (US Pat No. 4946778) can also be used to produce single chain antibodies against human pogo transposase 14.
  • Antibodies against human pogo transposase 14 can be used in immunohistochemical techniques to detect human pogo transposase 14 in biopsy specimens.
  • Monoclonal antibodies that bind to human pogo transposase 14 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • human pogo transposase 14 high-affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a sulfhydryl crosslinker such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human pogo transposase 14 positive cells .
  • the antibodies in the present invention can be used to treat or prevent diseases related to human pogotransposase 14. Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human pogo transposase 14.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human pogo transposase 14.
  • tests are well known in the art and include FI SH assays and radioimmunoassays.
  • the level of human pogo transposase 14 detected in the test can be used to explain the importance of human pogo transposase 14 in various diseases and to diagnose diseases in which human pogo transposase 14 plays 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.
  • the polynucleotide encoding human pogo transposase 14 can also be used for a variety of therapeutic purposes.
  • Gene therapy technology can be used to treat abnormal cell proliferation, development or metabolism caused by the non-expression or abnormal / inactive expression of human pogo transposase 14.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant human pogo transposase 14 to inhibit endogenous human pogo transposase 14 activity.
  • a mutated human pogo transposase 14 may be a shortened human pogo transposase 14 lacking a signaling domain, and although it can bind to a downstream substrate, it lacks signaling activity.
  • the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human pogotransposase 14.
  • Expression vectors derived from viruses such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding human pogo transposase 14 into a cell.
  • Methods for constructing recombinant viral vectors carrying a polynucleotide encoding human pogo transposase 14 can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human pogo transposase 14 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: injecting the polynucleotide directly into a tissue in vivo; or introducing the polynucleotide into a cell through a vector (such as a virus, phage, or plasmid) in vitro, The cells are then transplanted into the body and the like.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit human pogo transposase 14 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA, DM, 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 RNA.
  • This DNA sequence has been integrated downstream of the RNA polymerase promoter of the vector.
  • it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphorothioate or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human pogo transposase 14 can be used for the diagnosis of diseases related to human pogo transposase 14.
  • the polynucleotide encoding human pogo transposase 14 can be used to detect the expression of human pogo transposase 14 or the abnormal expression of human pogo transposase 14 in a disease state.
  • the DNA sequence encoding human pogo transposase 14 can be used to hybridize biopsy specimens to determine the expression of human pogo transposase 14.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are all mature and open technologies, and related kits are commercially available.
  • a part or all of the polynucleotides of the present invention can be used as probes for immobilization on a micro array (Mi c roa ray) or a DNA chip (also known as
  • Human pogo transposase 14 specific primers can also be used to detect the transcription products of human pogo transposase 14 by performing RNA-polymerase chain reaction (RT-PCR) in vitro amplification.
  • RT-PCR RNA-polymerase chain reaction
  • Detection of mutations in the human pogo transposase 14 gene can also be used to diagnose human pogo transposase 14-related diseases.
  • Human pogo transposase mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human pogo transposase 14 DNA sequence. Mutations can be detected using existing techniques such as Sou thern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, the Nort Hern blotting and Western blotting methods can be used to indirectly determine whether a gene is mutated.
  • sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, the specific loci of each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) can be used to mark chromosome locations. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.
  • PCR primers (preferably 15-35 bp) are prepared based on cDNA, which can be used to localize the sequence. Color body. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells that contain the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention by a similar method, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization (FI SH) of cDM clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FI SH 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, Mendelian Inherance in Man (available online with Johns Hopk ins Universe Wetch 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 of the affected individuals and the mutation is not observed in any normal individual, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the CDM that is 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 topical, intravenous, intraperitoneal, intramuscular, Subcutaneous, intranasal or intradermal route of administration.
  • Human pogo transposase 14 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human pogotransposase 14 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.
  • RNA Human fetal brain total 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.
  • the Smart cDNA cloning kit purchased from Clontech
  • the bacteria formed a cDNA library.
  • the sequences at the 5 'and 3' ends of all clones were determined using Dye terminate cyc l react ion sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer).
  • the determined cDNA sequence was compared with the existing public D sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0926b09 was new DNA.
  • the inserted cDNA fragments contained in this clone were determined in both directions by synthesizing a series of primers.
  • the sequence of the human pogo transposase 14 of the present invention and the protein sequence encoded by the human pogo transposase 14 were analyzed using the Blas t program (BasiclocalAlignment search tool) [Altschul, SF et al. J. Mol. Biol. 1990; 215: 403-10], perform homology search in databases such as Genbank, Swissport, etc.
  • the gene with the highest homology to the human pogo transposase 14 of the present invention is a known Drosophila transposase, and the accession number encoded by the protein in Genbank is 1149973.
  • the results of protein homology are shown in Figure 1. The two are highly homologous and their identity is 77%; the similarity is 85 «/ «.
  • Example 3 Cloning of a gene encoding human pogo transposase 14 by RT-PCR
  • CD was synthesized by reverse transcription reaction using fetal brain cell total RNA as a template and oligo-dT as a primer. After purification using Qiagene's kit, PCR was performed using the following primers:
  • Primer 1 5'- AGGTTGGTTCATGAGGTTTAAGG-3 '(SEQ ID NO: 3)
  • Primer2 5'- CACCTTATCAAATTTATTATTCC-3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Conditions for the amplification reaction 50 mmol / L KC1, 10 ramol / L Tris-Cl, (pH8.5), 1.5 mmol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol primers in a 50 ⁇ 1 reaction volume, 1U of 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 2min.
  • ⁇ -actin was set as a positive control and template blank was set as a negative control.
  • the amplified product was purified using a QIAGEN kit and ligated to a pCR vector (Invitrogen product) using a TA cloning kit. DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as l-2421bp shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of human pogo transposase 14 gene expression
  • This method involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue is homogenized with 4M guanidinium 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 ), Mix and 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.
  • RNA was prepared by random primer method using cx " 2 P dATP.
  • the D probe used was the PCR amplified human pogo transposase 14 coding region sequence (538bp to 930bp) shown in FIG.
  • 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 (pH 7.4)-5 x SSC- 5 x Denhardt's solution and 200 ⁇ g / ml salmon sperm DNA. After hybridization, the filter was washed in lx SSC-0.1% SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 5 In vitro expression, isolation and purification of recombinant human pogo transposase 14
  • Pr imer4 5,-CCCGGATCCCCGACTAAATCTTTAGGCCCCAC-3 '(Seq ID No: 6)
  • the 5' ends of these two primers contain Ncol and BamHI restriction sites, respectively, followed by the coding sequences of the 5 'and 3' ends of the target gene
  • the Ncol and BamHI restriction sites correspond to the selective endonuclease sites on the expression vector plasmid pET-28b (+) (product of Novagen, Cat. No. 69865. 3).
  • the PCR reaction was performed using the pBS-0926b09 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: 10 pg of pBS-0926b09 plasmid in a total volume of 50 ⁇ 1, Primer-3 and Primer-4 primers were 1 Opmol, Advantage polymerase Mi x (Clontech) 1 ⁇ 1, respectively. Cycle parameters: 94. C 20s, 60 ° C 30s, 68. C 2 min, a total of 25 cycles. Ncol and BamHI were used to double-digest the amplified product and plasmid P ET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into E. coli DH5c by the calcium chloride method.
  • a peptide synthesizer (product of PE company) was used to synthesize the following human pogo transposase 14-specific peptides: NH 2 -Met-Glu-Met-Tyr-Lys-Asp-I le-Asn-Va l-Ala-Phe- Met-Pro-Thr-Asn-COOH (SEQ ID NO: 7).
  • the polypeptide was coupled to hemocyanin and bovine serum albumin to form a complex. For the method, see: Avrameas, et al.

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Abstract

L'invention concerne un nouveau polypeptide, une pogo transposase humaine 14, et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la pogo transposase humaine 14.
PCT/CN2000/000353 1999-10-26 2000-10-23 Nouveau polypeptide, pogo transposase humaine 14, et polynucleotide codant pour ce polypeptide Ceased WO2001030835A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998040510A1 (fr) * 1997-03-11 1998-09-17 Regents Of The University Of Minnesota Systeme transposon a base d'adn permettant d'introduire de l'acide nucleique dans l'adn d'une cellule

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998040510A1 (fr) * 1997-03-11 1998-09-17 Regents Of The University Of Minnesota Systeme transposon a base d'adn permettant d'introduire de l'acide nucleique dans l'adn d'une cellule

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ROBERTSON H.M.: "Members of the pogo superfamily of DNA-mediated transposons in the human genome", MOL. GEN. GENET., vol. 252, October 1996 (1996-10-01), pages 761 - 766 *

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