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WO2001027283A1 - Nouveau polypeptide, proteine 16 de type transcriptase humaine inverse, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, proteine 16 de type transcriptase humaine inverse, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001027283A1
WO2001027283A1 PCT/CN2000/000313 CN0000313W WO0127283A1 WO 2001027283 A1 WO2001027283 A1 WO 2001027283A1 CN 0000313 W CN0000313 W CN 0000313W WO 0127283 A1 WO0127283 A1 WO 0127283A1
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Prior art keywords
polypeptide
protein
polynucleotide
reverse transcriptase
human reverse
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai BioDoor Gene Technology Ltd
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Shanghai BioDoor Gene Technology Ltd
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Priority to AU78984/00A priority Critical patent/AU7898400A/en
Publication of WO2001027283A1 publication Critical patent/WO2001027283A1/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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • 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 novel polypeptide, human reverse transcriptase-like protein 16, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a method and application for preparing the polynucleotide and polypeptide.
  • genetic recombination refers to the process of gene communication that causes genotypic changes. Genetic recombination is divided into four types: homologous recombination, site-specific recombination, transposition and abnormal recombination.
  • transposition in eukaryotes fall into two categories.
  • reposons Based on their structure and composition, reposons can be divided into viral and non-viral families.
  • reducing viruses In the virus family, the example of reducing viruses is used. Reverse transcription of infected viral RNA in the cytoplasm and synthesis and integration of double-stranded DNA are all catalyzed by reverse transcriptase. The specific mechanism has been clarified. In addition, the similarity between the reducing virus and the transposable element makes the reducing virus presumably evolved from the ancient transposable element. (Molecular Genetics, edited by Sun Naien, et al. Nanjing University Press 444-460)
  • LINE1 are a family of many copy number, a long sequence back seat element, PL 1 bad so long interspersed repetitive sequence 1 j (l ong interspersed repea ted sequences, LINEs / LINEl).
  • PL 1 bad so long interspersed repetitive sequence 1 j (l ong interspersed repea ted sequences, LINEs / LINEl).
  • LINE1 family is derived from a polynucleotide sequence encoding a reverse transcriptase-like protein. (Nature 1986 Jun 5-11; 321 (6070): 625-8)
  • HRTRP16 human reverse transcriptase-like protein 16
  • 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 human reverse transcriptase-like protein 16.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human reverse transcriptase-like protein 16.
  • Another object of the present invention is to provide a method for producing human reverse transcriptase-like protein 16.
  • Another object of the present invention is to provide antibodies against the polypeptide of the present invention, human reverse transcriptase-like protein 16.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors of the polypeptide of the present invention, human reverse transcriptase-like protein 16.
  • Another object of the present invention is to provide a method for diagnosing and treating a disease associated with abnormality of human reverse transcriptase-like protein 16.
  • a novel isolated human reverse transcriptase-like protein 16 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, the nucleotide sequence having at least 69 with a nucleotide sequence selected from the group consisting of % Identity: (a) a polynucleotide encoding the aforementioned human reverse transcriptase-like protein 16; (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 704-1 1 at 35 in SEQ ID NO: 1; and (b) having a sequence 1 at SEQ ID NO: 1 -1503 bit sequence.
  • Fig. 1 is a comparison diagram of the amino acid sequence homology of human reverse transcriptase-like protein 16 and human reverse transcriptase-related protein of the present invention.
  • the upper sequence is human reverse transcriptase-like protein 16 and the lower sequence is human reverse transcriptase-related protein.
  • Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+”.
  • Figure 1 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of an isolated human reverse transcriptase-like protein. 16kDa 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 existing in the natural state. .
  • isolated means that human reverse transcriptase-like protein 16 is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify human reverse transcriptase-like protein 16 using standard protein purification techniques. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the human reverse transcriptase-like protein 16 peptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human reverse transcriptase-like protein 16, 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 polypeptide of the present invention may be a naturally purified product or a chemically synthesized product, or may be produced from a prokaryotic or eukaryotic host (for example, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant technology. 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 reverse transcriptase-like protein 16.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human reverse transcriptase-like protein 16 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 left over Codon-encoded; or ( ⁇ ) such a type in which a group on one or more amino acid residues is substituted with another group to include a substituent; or ( ⁇ ⁇ ) such a type in which the mature polypeptide and Fusion of another compound (such as a compound that extends the half-life of a polypeptide, such as polyethylene glycol); or (IV) a polypeptide sequence (such as a leader sequence or a secreted sequence or The sequences used to purify this polypeptide or protease sequences) As set forth 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 CDM library of human fetal brain tissue. It contains a 1503 base polynucleotide sequence, and its open reading frame (704-11 35) encodes 143 amino acids. According to the amino acid sequence homology comparison, it was found that the polypeptide has 70% homology with the human reverse transcriptase-related protein.
  • the polynucleotide of the present invention may be in the form of DM or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the DM can be a coding chain or a non-coding chain.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant” refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide that includes the polypeptide and a polynucleotide that includes additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • This polynucleotide variant can be a naturally occurring allelic variant or a non-naturally occurring variant.
  • These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity between the two sequences).
  • the invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions.
  • “strict conditions” means: (1) at lower ionic strength and Hybridization and elution at high temperature, such as 0.2xSSC, 0.1% SDS, 6 (TC; or (2) with denaturants such as 50% (v / v) formamide, 0.1% calf serum / 0 l% Ficoll, 42 ° C, etc .; or (3) hybridization occurs only when the identity between the two sequences is at least 95% or more, and more preferably 97% or more.
  • the hybridizable polynucleotide encodes The polypeptide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nuclei. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding human reverse transcriptase-like protein 16.
  • 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 reverse transcriptase-like protein 16 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDNA of interest is to isolate 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 polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determining the level of human reverse transcriptase-like protein 16 transcripts; ( 4) Detecting gene-expressed protein products by immunological techniques or by measuring biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probes used here are typically the genes of the invention Sequence information is based on chemically synthesized DM sequences. The genes or fragments of the present invention can of course be used as probes. DM probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product of human reverse transcriptase-like protein 16 gene expression.
  • ELISA enzyme-linked immunosorbent assay
  • a method using PCR to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-cDM terminal rapid amplification method
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be measured by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDM sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDM sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a human reverse transcriptase-like protein 16 coding sequence, and a recombinant technology for producing the polypeptide of the present invention. method.
  • a polynucleotide sequence encoding a human reverse transcriptase-like protein 16 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors expressed in bacteria (Rosenberg, et al.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • DM sequences encoding human reverse transcriptase-like protein 16 and appropriate transcription / translation regulatory elements can be used to construct expression vectors containing DM sequences encoding human reverse transcriptase-like protein 16 and appropriate 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 DM 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 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 expressed by DM, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers 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 a human reverse transcriptase-like protein 16 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form 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 s melanoma cells, etc. .
  • Transformation of a host cell with a DNA sequence according to the present invention or a recombinant vector containing the DM sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of DNA uptake can be in the exponential growth phase were harvested, treated with (Method 12, using the procedure well known in the art.
  • Alternative is MgC l 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.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human reverse transcriptase-like protein 16 (Scence, 1984; 224: 14 31). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid
  • the polypeptide of the present invention is closely related to the process of transposition, especially to the process of non-viral return.
  • AI DS human acquired immune deficiency syndrome
  • the strong oncovirus in the reduced virus contains an oncogene (one), which can rapidly induce tumors in animals, and the similarity between the reduced virus and transposons and retroposons makes the polypeptide of the present invention inferred from The occurrence of tumors has a lot to do with it.
  • the polypeptide of the present invention can be used for the diagnosis and treatment of malignant tumors, including leukemias and lymphomas; tumors of epithelial cell origin; tumors of mesenchymal origin, such as sarcomas; central nervous system tumors and the like.
  • the polypeptides and antibodies of the present invention also have effects on damage, defects or disorders of immune tissues, especially for diseases of the hematopoietic system (such as malignant anemia), skin diseases (such as psoriasis), and autoimmune diseases (such as rheumatoid arthritis). ), Radiation diseases and the production and regulation of immune lymphocytes are extremely closely related.
  • diseases of the hematopoietic system such as malignant anemia
  • skin diseases such as psoriasis
  • autoimmune diseases such as rheumatoid arthritis
  • the invention also provides methods of screening compounds to identify agents that increase (agonist) or suppress (antagonist) human reverse transcriptase analogue protein 16.
  • Agonists enhance biological functions such as human reverse transcriptase-like protein 16 to stimulate cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing human reverse transcriptase-like protein 16 can be cultured with labeled human reverse transcriptase-like protein 16 in the presence of drugs. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human reverse transcriptase-like protein 16 include selected antibodies, compounds, receptor deletions, and the like. Antagonists of human reverse transcriptase-like protein 16 can bind to human reverse transcriptase-like protein 16 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 function biologically. Learn function.
  • human reverse transcriptase-like protein 16 can be added to bioanalytical assays to determine whether a compound is a compound by measuring the effect of the compound on the interaction between human reverse transcriptase-like protein 16 and its receptor. Antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same way as for screening compounds described above.
  • Polypeptide molecules capable of binding to human reverse transcriptase-like protein 16 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 reverse transcriptase-like protein 16 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 directed against human reverse transcriptase-like protein 16 epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments generated from Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human reverse transcriptase-like protein 16 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 Agent.
  • Techniques for preparing monoclonal antibodies against human reverse transcriptase-like protein 16 include, but are not limited to, hybridoma technology (Kohler and Mil te in. Nature, 1975, 256: 495-497), triple tumor technology, human beta- Cell hybridoma technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions and non-human variable regions can be produced using existing techniques (Morr et al, PNAS, 1985, 81: 6851). 0 Existing techniques for producing single-chain antibodies (US Pa t No. 4946778) can also be used to produce single chain antibodies against human reverse transcriptase-like protein 16.
  • Anti-human reverse transcriptase-like protein 16 antibodies can be used in immunohistochemistry to detect human reverse transcriptase-like protein 16 in biopsy specimens.
  • Monoclonal antibodies that bind to human reverse transcriptase-like protein 16 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. For example, a human reverse transcriptase-like protein 16 with high affinity can be covalently bound to bacterial or phytotoxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human reverse transcriptase-like protein 16 positive Cell.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human reverse transcriptase-like protein 16.
  • Administration of an appropriate dose of antibody can stimulate or block the production or activity of human reverse transcriptase-like protein 16.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human reverse transcriptase-like protein 16 levels. These tests are well known in the art and include F I SH assays and radioimmunoassays.
  • the level of human reverse transcriptase-like protein 16 detected in the test can be used to explain the importance of human reverse transcriptase-like protein 16 in various diseases and to diagnose diseases in which human reverse transcriptase-like protein 16 functions .
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • Polynucleotides encoding human reverse transcriptase-like protein 16 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 reverse transcriptase-like protein 16.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant human reverse transcriptase-like protein 16 to inhibit endogenous human reverse transcriptase-like protein 16 activity.
  • a variant human reverse transcriptase-like protein 16 may be shortened and lack human signal transduction domain-like human reverse transcriptase-like protein 16. Although it can bind to downstream substrates, it lacks signal transduction activity.
  • the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human reverse transcriptase-like protein 16.
  • 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 human reverse transcriptase-like protein 16 into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a human reverse transcriptase-like protein 16 can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human reverse transcriptase-like protein 16 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit human reverse transcriptase-like protein 16 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA component that specifically breaks down specific RMs. The mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA and performs endonucleation.
  • Antisense RM, DM, 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 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 vector's RNA polymerase promoter. 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.
  • the polynucleotide encoding human reverse transcriptase-like protein 16 can be used for the diagnosis of diseases related to human reverse transcriptase-like protein 16.
  • the polynucleotide encoding human reverse transcriptase-like protein 16 can be used to detect the expression of human reverse transcriptase-like protein 16 or the abnormal expression of human reverse transcriptase-like protein 16 in a disease state.
  • a DNA sequence encoding human reverse transcriptase-like protein 16 can be used to hybridize biopsy specimens to determine the expression of human reverse transcriptase-like protein 16.
  • Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and so on. These techniques and methods are publicly available and mature, and the relevant kits are commercially available.
  • a part or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also called a "gene chip") for analyzing differential expression analysis of genes and genetic diagnosis in tissues.
  • Human reverse transcriptase-like protein 16 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect human reverse transcriptase-like protein 16 transcription products.
  • Detection of mutations in the human reverse transcriptase-like protein 16 gene can also be used to diagnose human reverse transcriptase-like protein 16-related diseases.
  • Human reverse transcriptase-like protein 16 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type human reverse transcriptase-like protein 16 DNA sequences. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, the specific loci of each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) can be used to mark chromosome locations. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared according to CDM, and the sequences can be mapped on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells that contain the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention by a similar method, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendel ian Inheritance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine 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 reverse transcriptase-like protein 16 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dosage range of human reverse transcriptase-like protein 16 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.
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using Quik mRNA I solat ion Kit (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA.
  • the Smart cDNA cloning kit purchased from Clontech was used to insert the cDNA fragment into the multiple cloning site of the pBSK (+) vector (Clontech) to transform DH5 ⁇ .
  • the bacteria formed a cDNA library.
  • the sequences at the 5 'and 3' ends of all clones were determined using Dye terminate cyc le react ion sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer).
  • the determined cDNA sequence was compared with an existing public DM sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0210b09 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • the 0210b09 clone contains a full-length cDNA of 1503bp (as shown by Seq ID N0: 1), and a 432bp open reading frame (0RF) from 704bp to 1135bp, encoding a new protein (such as Seq ID NO : Shown in 2).
  • This clone P BS-0210b09 and the encoded protein was named human reverse transcriptase-like protein 16.
  • Example 2 Homologous search of cDNA clones
  • the human reverse transcriptase-like protein 16 sequence of the present invention and its encoded protein sequence were analyzed using the Blas t program (Basiclocal Alignment search tool) [Altschul, SF et al. J. Mol. Biol. 1990; 215: 403-10 ], To perform homology search in databases such as Genbank and Swiss sport.
  • the gene with the highest homology to the human reverse transcriptase-like protein 16 of the present invention is a known human reverse transcriptase-related protein gene.
  • the protein homology result is shown in FIG. 1, and the two are highly homologous. 70%; Similarity is 81%.
  • Example 3 Cloning of a gene encoding human reverse transcriptase-like protein 16 by RT-PCR
  • CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, the following primers were used for PCR amplification:
  • Primer 1 5'-AGATAGAGACACTGAAAACTCTTCCAAAAA-3 '(SEQ ID NO: 3)
  • Primer2 5'-AAGGTTGGCTTGGCTATTTGGGCTCTTTT-3' (SEQ IDNO: 4)
  • Priraerl is a forward sequence starting at the 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 ⁇ l of KC1, 10 mmol / L Tris-Cl, (pH8.5), 1.5 mmol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol in a reaction volume of 50 ⁇ 1 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 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 using a TA cloning kit (Invitrogen).
  • the DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as the 1-1503bp shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of human reverse transcriptase-like protein 16 gene expression Total RM was extracted by a one-step method [Anal. Biochem 1987, 162, 156-159]. This method involves acid guanidinium thiocyanate-chloroform extraction. i.e.
  • RNA precipitate wash the obtained RNA precipitate with 70% ethanol, dry and dissolve in water.
  • RNA probe was the sequence of the human reverse transcriptase-like protein 16 coding region (704bp to 1135bp) amplified by PCR as shown in FIG. 1 ).
  • a pair of specific amplification primers is designed, and the sequences are as follows: Primer3: 5'-CCCCCATGGATGATTATCTCAATAAATGCAG-3 '(Seq ID No: 5) Priraer4: 5'-CCCGGATCCCTACTTTTGCAGATTGATTTTA-3' (Seq ID No: 6) The 5 'ends of these two primers contain Ncol and BamHI restriction sites, respectively. The coding sequences of the 5 'and 3' ends of the gene of interest are followed, respectively.
  • the Ncol and BamHI restriction sites correspond to the selectivity within the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Digestion site.
  • the PCR reaction was performed using the pBS-0210b09 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of pBS-0210b09 plasmid, primers Primer-3 and Primer-4 were 1 Opmol, Advantage polymerase Mix (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 pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligation product was transformed into E. coli DH5a by the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 30 g / ml), positive clones were screened by colony PCR method and sequenced. A positive clone (pET-0210b09) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method.
  • a peptide specific to human reverse transcriptase-like protein 16 was synthesized using a peptide synthesizer (product of PE): NH2-Met-I le-I le-Ser-I le-Asn-Ala-Glu-Lys-Ala-Ser -Asn-Lys-I le-Gln-0H (SEQ ID NO: 7).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once. ⁇ Using a 15 g / ml bovine serum albumin peptide complex-coated titer plate as an ELISA to determine antibody titers in rabbit serum. Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose. The peptide was bound to a cyanogen bromide-activated Seph a rose 4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography. The immunoprecipitation method proved that the purified antibody could specifically bind to human reverse transcriptase-like protein 16.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine 16 de type transcriptase humaine inverse, et polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes par ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment les tumeurs malignes, l'hémopathie, l'infection par VIH, les maladies immunitaires et diverses inflammations. L'invention concerne aussi les agonistes agissant contre le polypeptide et leur action thérapeutique ainsi que les applications de ce polynucléotide codant pour la nouvelle protéine 16 de type transcriptase humaine inverse.
PCT/CN2000/000313 1999-10-13 2000-10-12 Nouveau polypeptide, proteine 16 de type transcriptase humaine inverse, et polynucleotide codant pour ce polypeptide Ceased WO2001027283A1 (fr)

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AU78984/00A AU7898400A (en) 1999-10-13 2000-10-12 A novel polypeptide, a human reverse transcriptase like protein 16 and the polynucleotide encoding the polypeptide

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CN99116963 1999-10-13
CN99116963.8 1999-10-13

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK *
GENOME RES., vol. 8, no. 11, November 1998 (1998-11-01), pages 1097 - 1108 *

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