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WO2002004503A1 - Nouveau polypeptide, proteine humaine 15 associee a la transcription inverse orf2 du facteur l1, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, proteine humaine 15 associee a la transcription inverse orf2 du facteur l1, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002004503A1
WO2002004503A1 PCT/CN2001/000981 CN0100981W WO0204503A1 WO 2002004503 A1 WO2002004503 A1 WO 2002004503A1 CN 0100981 W CN0100981 W CN 0100981W WO 0204503 A1 WO0204503 A1 WO 0204503A1
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Prior art keywords
polypeptide
human
polynucleotide
reverse transcription
factor
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PCT/CN2001/000981
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
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Application filed by Biowindow Gene Development Inc. Shanghai filed Critical Biowindow Gene Development Inc. Shanghai
Priority to AU93619/01A priority Critical patent/AU9361901A/en
Publication of WO2002004503A1 publication Critical patent/WO2002004503A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology, specifically, the present invention describes a new polypeptide One human L1 factor ORF2 reverse transcription-related protein 15 and a polynucleotide sequence encoding the polypeptide.
  • the invention also relates to methods and applications for preparing such polynucleotides and polypeptides. Background technique
  • the primate and rodent genomes contain an L1 family with highly repetitive, long, and alternating sequences.
  • the sequence of this family is characterized by an A-rich sequence at the 3'-terminus and the 5, -terminus. It is a blunt-end structure, which is different from the general sticky-end structure, but does not affect its transcription integration process, and has longer open reading frames (ORFs).
  • ORFs open reading frames
  • Members of the L1 family exist in different types of cells, including multifunctional embryonic cells, which proves that the L1 family originates from the same sequence coding protein and is dispersed into the genome through an RNA-mediated process.
  • the L1 family is involved in the reverse transcription process. The reverse transcription starts at the 3'-terminus and ends at overlapping sites caused by complementary DNA inserts. It can be converted into cDNA through transcription, and then inserted into a process similar to pseudogene processing In the chromosome.
  • the L1 factor is present on LRE2 on chromosome lq. Structurally, the L1 factor does not have a long terminal repeat. The 3, -terminal poly A tail is special, and it is not fixed on each L1 factor.
  • the proliferation process of the L1 factor includes transcription, reverse transcription, and reintegration into a new site on the genome. Most of the L1 factor's 5 ends are blunt-end structures, which are different from the general sticky-end structure, but do not affect its transcription integration process.
  • the untranslated region (UTR) at the 5 'end contains an internal promoter that regulates and controls the speed of the entire reverse transcription process.
  • the LRE2 site contains two complete open reading frames (0RF1 and 0RF2).
  • the 0RF1 frame encodes an approximately 40Kd embryonic cancer cell protein.
  • the 0RF2 frame is sequenced with reverse transcriptase and other retroviral proteins. Similarity can mediate the process of reverse transcription. Using experimental methods to remove the first 32Bp of the L1 factor, the obtained product can only show 20% of the normal transcription efficiency.
  • L1 family sequence An open reading frame of the L1 family sequence is similar to some RNA-dependent DM polymerases of viruses. It further proves that the L1 family or its ancestors are related to reverse transcriptase, and the protein it encodes is actually a class Reverse transcriptase.
  • the polypeptide of the present invention has 56 »/ in the ORF2 region of the aforementioned L1 factor. Identity, 71% similarity, and contains its characteristic conserved sequences, so this protein is considered to be a new L1 factor 0RF2 phase Related proteins have similar biological functions and are named human L1 factor 0RF2 reverse transcription-related protein 15. As described above, the human L1 factor 0RF2 reverse transcription-related protein 15 protein plays an important role in regulating important functions of the body, such as cell division and embryo development, and it is believed that a large number of proteins are involved in these regulatory processes, so the identification of Most of the human L1 factor 0RF2 reverse transcription-related protein 15 proteins involved in these processes, especially the amino acid sequence of this protein.
  • Isolation of the novel human L1 factor 0RF2 reverse transcription-related protein 15 protein encoding gene also provides a basis for research to determine the role of this protein in health and disease states.
  • This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding for DM. Disclosure of invention
  • 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 the human L1 factor ORF2 reverse transcription-related protein 15.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding the human L1 factor ORF2 reverse transcription-related protein 15.
  • Another object of the present invention is to provide a method for producing human L1 factor ORF2 reverse transcription-related protein 15. Another object of the present invention is to provide an antibody against the polypeptide of the present invention-human L1 factor ORF2 reverse transcription-related protein 15.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors of the polypeptide of the present invention-human L1 factor ORF2 reverse transcription-related protein 15.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormality of human L1 factor 0RF2 reverse transcription-related protein 15.
  • the present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 914-1321 in SEQ ID NO: 1; and (b) a sequence having 1-2160 in SEQ ID NO: 1 Sequence of bits.
  • the present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit human L1 factor 0RF2 reverse transcription related protein 15 protein activity, which comprises utilizing the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for detecting a disease or disease susceptibility related to abnormal expression of human L1 factor 0RF2 reverse transcription-related protein 15 protein in vitro, comprising detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample. Or detecting the amount or biological activity of a polypeptide of the invention in a biological sample.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of human L1 factor ORF2 reverse transcription related protein 15 .
  • Nucleic acid sequence refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and can also refer to genomic or synthetic DNA or RNA, which can be single-stranded or double-stranded, representing the sense strand or Antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and a fragment or part thereof.
  • a protein or polynucleotide “variant” refers to an amino acid sequence having one or more amino acids or nucleotide changes, or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. Variants can have "conservative" changes, in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as the replacement of Leucine. Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response in appropriate animals or cells and to bind to specific antibodies.
  • An "agonist” refers to a molecule that, when combined with human L1 factor 0RF2 reverse transcription-related protein 15, can cause the protein to change, thereby regulating the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that binds human L1 factor ORF2 reverse transcription-related protein 15.
  • Antagonist refers to a biological or immunological activity that can block or regulate human L1 factor 0RF2 reverse transcription-related protein 15 when combined with human L1 factor 0RF2 reverse transcription-related protein 15 Molecule.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that binds human L1 factor 0RF2 reverse transcription-related protein 15.
  • “Regulation” refers to a change in the function of human L1 factor 0RF2 reverse transcription-related protein 15, including an increase or decrease in protein activity, changes in binding characteristics, and any other biological properties of human L1 factor 0RF2 reverse transcription-related protein 15 , Functional or immune properties.
  • substantially pure is meant substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify human L1 factor 0RF2 reverse transcription-related protein 15 using standard protein purification techniques.
  • the substantially pure human L1 factor 0RF2 reverse transcription-related protein 15 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of human L1 factor 0RF2 reverse transcription-related protein 15 peptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence C-T-G-A
  • complementary sequence G-A-C-T.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. The inhibition of such hybridization can be detected by performing hybridization (Southern blotting or Nor thern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit Binding of a homologous sequence to a target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences.
  • the percentage identity can be determined electronically, such as by the MEGALIGN program (Laser gene software package, DNASTAR, Inc., Mad Son Wis.).
  • the MEGALIGN program can compare two or more sequences according to different methods, such as the Clus ter method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). 0
  • the Clus ter method groups each group by checking the distance between all pairs. The sequences are arranged in clusters. The clusters are then assigned in pairs or groups.
  • the percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: The number of matching residues between sequence A and sequence X 100 The number of residues in sequence A-the number of spacer residues in sequence A The number of spacer residues in a sequence B can also be determined by Clus ter method or using methods known in the art such as Jotun Hein.
  • the percent identity between nucleic acid sequences (Hein J., (1990) Methods in emzumol ogy 183: 625-645 ) 0 "Similarity" refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitutions may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DM or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? (& 13 ') 2 and? ⁇ It can specifically bind to the epitope of human L1 factor ORF2 reverse transcription-related protein 15.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of matter from its original environment (for example, Natural environment).
  • a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not a component of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances in the natural state .
  • isolated human L1 factor 0RF2 reverse transcription-related protein 15 refers to human L1 factor 0RF2 reverse transcription-related protein 15 which is substantially free of other proteins, lipids, carbohydrates, or other substances naturally associated with it.
  • Those skilled in the art can purify human L1 factor ORF2 reverse transcription-related protein 15 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human L1 factor 0RF2 reverse transcription-related protein 15 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a novel polypeptide, namely human L1 factor 0RF2 reverse transcription-related protein 15, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of human L1 factor ORF2 reverse transcription-related protein 15.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human L1 factor ORF2 reverse transcription-related protein 15 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 the genetic code; or (II) such a type in which a group on one or more amino acid residues is substituted by other groups to include a substituent; or (III) such One, in which the mature polypeptide is fused to another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV) such a polypeptide sequence in which the additional amino acid sequence is fused into the mature polypeptide ( As described herein, such fragments, derivatives and analogs are considered as leader sequences or secreted sequences or sequences used to purify this polypeptide. It is 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 SBQ ID NO: 1.
  • the polynucleotide of the present invention is found from a CDM library of human fetal brain tissue. It contains a full-length polynucleotide sequence of 2160 bases, and its open reading frame 914-1321 encodes 135 amino acids. According to the amino acid sequence homology comparison, it was found that this polypeptide has 56% homology with human L1 factor 0RF2 reverse transcription-related protein. It can be concluded that the human L1 factor 0RF2 reverse transcription-related protein 15 has human L1 factor 0RF2 reverse transcription Similar structures and functions of related proteins.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DM.
  • DM can be single-stranded or double-stranded.
  • 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 having a sequence 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 naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity between the two sequences).
  • the invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions.
  • stringent conditions means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1 »/. SDS, 60 ° C; or (2) adding a denaturant during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1 ° /.
  • hybridization occurs only when the identity between the two sequences is at least 95%, and more preferably 97%.
  • polypeptide encoded by the hybridizable polynucleotide is identical to the mature polypeptide shown in SEQ ID NO: 2 Biological function and activity.
  • 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 L1 factor 0RF2 reverse transcription-related protein 15. '
  • 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 L1 factor ORF2 reverse transcription-related protein 15 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DM fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the MA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DM is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the separation of cMA sequences.
  • the standard method for isolating the cMA 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.
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manua, 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.
  • the genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (1) DM-MA or MA-RM hybridization; (2) the appearance or loss of marker gene function; (3) determination of the transcript of human L1 factor 0RF2 reverse transcription-related protein 15 Level; (4) detecting protein products of gene expression by immunological techniques or measuring biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used herein is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention. The genes or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product of human L1 factor 0RF2 reverse transcription-related protein 15 gene expression.
  • ELISA enzyme-linked immunosorbent assay
  • a method using PCR technology to amplify DNA / RM (Saiki, et al. Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified MA / MA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DM fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, the sequencing must be repeated. Sometimes it is necessary to determine the cDM sequences of multiple clones in order to splice into full-length cDM sequences.
  • 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 human L1 factor ORF2 reverse transcription related protein 15 coding sequence, and the recombinant technology to produce the present invention Said method of polypeptide.
  • a polynucleotide sequence encoding the human L1 factor 0RF2 reverse transcription-related protein 15 may be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (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 L1 factor ORF2 reverse transcription-related protein 15 and appropriate transcription / translation regulatory elements can be used to construct expression vectors containing DM sequences encoding human L1 factor ORF2 reverse transcription-related protein 15 and appropriate transcription / translation regulatory elements. These methods include in vitro recombination DM technology, DM synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manua, cold Spring Harbor Laboratory. New York, 1989).
  • the MA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRM synthesis. Representative examples of these promoters are: the lac 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 at a later stage of the origin of replication, polyoma enhancers and adenovirus enhancers at 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 the human L1 factor 0RF2 reverse transcription-related protein 15 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a genetic engineering containing the polynucleotide or the recombinant vector.
  • 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 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 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 following DNA 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 L1 factor 0RF2 reverse transcription-related protein 15 (Sc ience, 1984; 224: 1431). Generally speaking, 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.
  • the physical, chemical, and other properties can be used to separate and purify the recombinant protein by various separation methods. 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
  • Fig. 1 is a comparison diagram of amino acid sequence homology of the inventor's L1 factor 0RF2 reverse transcription-related protein 15 and human L1 factor 0RF2 reverse transcription-related protein.
  • the upper sequence is human L1 factor 0RF2 reverse transcription-related protein 15, and the lower sequence is human L1 factor 0RF2 reverse transcription-related protein.
  • 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 L1 factor 0RF2 reverse transcription-related protein 15. 15kDa is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention
  • the determined cDNA sequence was compared with the existing public DM sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 1700c05 was new DNA.
  • a series of primers were synthesized to perform bidirectional determination of the inserted CDM fragments contained in this clone.
  • the results show that the full-length cDNA contained in the 1700c05 clone is 2160bp (as shown in Seq ID NO: l), and there is a 408bp open reading frame (0RF) from 914bp to 1321bp, which encodes a new protein (such as Seq ID NO : Shown in 2).
  • This clone P BS-1700c05 and the encoded protein was named human L1 factor 0RF2 reverse transcription-related protein 15.
  • Example 2 Homologous search of cDNA clones
  • the sequence of the human L1 factor ORF2 reverse transcription-related protein 15 of the present invention and the protein sequence encoded by the same are used by the Blast program (BasiclocalAlignity search tool) [Altschul, SF et al. J. Mol. Biol. 1990; 215: 403-10], perform homology search in databases such as Genbank and Swissport.
  • the gene with the highest homology to the human L1 factor 0RF2 reverse transcription-related protein 15 of the present invention is a known human LI factor 0RF2 reverse transcription-related protein, and its encoded protein has the accession number M80341 in Genbank.
  • the protein homolog results are shown in Figure 1. The two are highly homologous, with an identity of 56% and a similarity of 71%.
  • Example 3 Cloning of a gene encoding human L1 factor 0RF2 reverse transcription-related protein 15 by RT-PCR
  • CDM was synthesized using fetal brain cell total MA as a template and ol igo-dT as a primer for reverse transcription reaction.
  • PCR amplification was performed with the following primers:
  • Pr imer 1 5'- AGACATGGGAGAGACAGGCAAGCT-3, (SEQ ID NO: 3)
  • Pr imer 2 5,-CATAGGCCGAGGCGGCCGACATGT -3, (SEQ ID NO: 4)
  • Pr imerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Pr imer2 is the 3'-end reverse sequence in SEQ ID NO: 1.
  • Amplification reaction conditions 50-leg ol / L KC1, 10-leg ol / L Tris-Cl, ( ⁇ 8.5), 1.5 mmol / L MgCl 2 , 200 ⁇ mol in a reaction volume of 50 ⁇ 1 / L dNTP, lOpmo l primer, 1U Taq DNA polymerase (Clontecli).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72. C 2min.
  • RT-PCR set ⁇ -act in as a positive control and template blank as a negative control.
  • the amplified product was purified using a QIAGEN kit and ligated to a pCR vector (Invitrogen) using a TA cloning kit.
  • the DNA sequence analysis results showed that the DM sequence of the PCR product was exactly the same as 1-2160bp shown in SBQ ID NO: 1.
  • Example 4 Analysis of human LI factor ORF2 reverse transcription-related protein 15 gene expression by Northern blotting method: Total MA was extracted in one step [Ana l. Biochem 1987, 162, 156-159]. This method involves acid guanidinium thiocyanate phenol-chloroform extraction.
  • the tissue was homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1), centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The obtained MA precipitate was washed with 70% ethanol, dried and dissolved in water. With 20 g RM, electrophoresis was performed on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (PH7.0)-5raM sodium acetate-ImM EDTA-2.
  • a 32 P dATP was used to prepare 32 P-labeled DNA probes by random primers.
  • the DNA probe used was the PCR-encoded human L1 factor ORF2 reverse transcription-related protein 15 coding region sequence (914bp to 1321bp) shown in FIG. 1.
  • 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-25 ⁇ M KH 2 P0 4 ( ⁇ 7.4)-5 x SSC-5 x Denhardt's solution and 20 ( ⁇ g / ml salmon sperm DM.
  • Example 5 In vitro expression, isolation and purification of recombinant human L1 factor ORF2 reverse transcription-related protein 15
  • Pr imer 3 5 '-CATGCTAGCATGCTGGAAAAGCACTTGATAAAA- 3' (Seq ID No: 5)
  • Pr imer4 5'-CATGGATCCTTATAGGTTGATTTTGTATCCTGC-3 '(Seq ID No: 6)
  • the 5' ends of these two primers contain Nhel and BamHI digestion respectively Site, followed by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively.
  • the Nhel and BamHI restriction sites correspond to the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865. 3) Selective endonuclease site.
  • PCR was performed using the pBS-1700c05 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-1700c05 plasmid, primers Primer-3 and Primer- 4 points; j is l Opmol, Advantage polymerase Mix (Clontecli) 1 ⁇ 1. Cycle parameters: 94 ° C 20s, 60. C 30s, 68 ° C 2 min, a total of 25 cycles. Nhel 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 the colibacillus DH5 cx by the calcium chloride method. After being cultured overnight on an LB plate containing kanamycin (final concentration 30 g / ml), positive clones were selected by colony PCR method and sequenced. A positive clone (pET-1700b05) 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. In LB liquid medium containing kanamycin (final concentration 30 ⁇ g / ml), the host strain BL (pET-1700c05) was at 37.
  • NH2-Met-Leu-Glu-Lys-His-Leu-I le-Lys-Phe-Asn-I le-Leu-Ser-Cys-Leu- COOH SEQ ID NO: 7
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin for methods, see: Avrameas, et al. Immunochemistry, 1969; 6: 43. Rabbits were immunized with 1 ⁇ 2 g of the hemocyanin peptide complex plus complete Freund's adjuvant; 15 days later, the hemocyanin peptide complex plus incomplete Freund's adjuvant was used to boost immunity once.
  • a titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum.
  • Protein A-Sepharose was used to isolate total IgG from antibody-positive rabbit sera.
  • the peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography.
  • the immunoprecipitation method proved that the purified antibody could specifically bind to human L1 factor 0RF2 reverse transcription-related protein 15.
  • Example 7 Application of the polynucleotide fragment of the present invention as a hybridization probe
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select suitable oligonucleotide fragments from the polynucleotide SBQ ID NO: 1 of the present invention as hybridization probes, and to identify whether some tissues contain the polynucleosides of the present invention using a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods. They all use the same steps of hybridization after fixing the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer so that the non-specific binding site of the sample on the filter is loaded • Saturated by polymers and synthetic polymers.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid.
  • the probes from the hybridization are removed by a series of membrane washing steps.
  • This embodiment utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under high-intensity washing conditions, the specificity of the hybridization of the first type of probe with the sample is the strongest and is retained.
  • oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • the GC content is 30% -70%, and the non-specific hybridization increases when it exceeds;
  • the preliminary selection probe and its source sequence region ie, SEQ ID NO: 1
  • other known genomic sequences and their The complementary region is compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutant sequence (41Nt) of the gene fragment of SEQ ID NO: 1 or its complementary fragment:
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membrane nitrocellulose membrane
  • the sample membrane was placed in a plastic bag, and 3-10 mg of prehybridization solution (1 OxDenhardf s; 6xSSC, 0.1 mg / ml) was added.
  • CT DNA (calf thymus DNA).
  • polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infections and immune diseases. .
  • the human genome contains an L1 family whose sequence features include the presence of long open reading frames (ORFs).
  • ORFs long open reading frames
  • Members of the L1 family exist in different cell types, including multifunctional embryonic fetal cells.
  • the L1 family is involved in the reverse transcription process, which can be converted into cDM through transcription and then inserted into the chromosome through a process similar to pseudogene processing.
  • the L1 factor is present on LRE2 of chromosome lq, and the LRE2 locus contains two complete open reading frames (0RF1 and 0RF2), of which the 0RF2 frame is related to reverse transcriptase and other retroviral proteins Sequence similarity can mediate the reverse transcription process.
  • polypeptides of the present invention are highly homologous to the ORF2 type reverse transcriptase of the human L1 factor, and contain the characteristic sequences of the ORF2 type reverse transcriptase family of the L1 factor, both of which have similar biological functions. It is involved in certain reverse transcription processes in the body and the insertion and dissemination of genes in chromosomes, which is important for genetic variation. Its abnormal expression is usually closely related to the occurrence of some related disorders of substance metabolism, disorders of protein metabolism, and tumors and cancers of related tissues, and produce related diseases.
  • abnormal expression of the human L1 factor 0RF2 reverse transcription-related protein 15 of the present invention will produce Various diseases, especially various tumors, embryonic developmental disorders, growth disorders, inflammation, immune diseases, these diseases include, but are not limited to:
  • Tumors of various tissues stomach cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioblastoma, nerve Fibroma, colon cancer, melanoma, bladder cancer, uterine cancer, endometrial cancer, colon cancer, thymic tumor, nasopharyngeal cancer, laryngeal cancer, tracheal tumor, fibroid, fibrosarcoma, lipoma, liposarcoma
  • Fetal developmental disorders congenital abortion, cleft palate, limb loss, limb differentiation disorder, atrial septal defect, neural tube defect, congenital hydrocephalus, congenital glaucoma or cataract, congenital deafness, growth and development disorders: mental development Retardation, brain development disorders, skin, fat, and muscular dysplasia, bone and joint dysplasia, various metabolic deficiencies, stunting, dwarfism, Cushing's syndrome, sexual retardation
  • Inflammation chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, cerebrospinal multiple sclerosis, glomerulonephritis, myocarditis, cardiomyopathy, atherosclerosis, gastric ulcerative cervicitis, each Infectious inflammation
  • Immune diseases Systemic lupus erythematosus, rheumatoid arthritis, bronchial asthma, urticaria, specific dermatitis, post-infection myocarditis, scleroderma, myasthenia gravis, Guillain-Barre syndrome, common variable immunodeficiency disease , Primary B-lymphocyte immunodeficiency disease, Acquired immunodeficiency syndrome
  • the abnormal expression of the human L1 factor 0RF2 reverse transcription-related protein 15 of the present invention will also produce certain hereditary, hematological diseases and the like.
  • polypeptides of the present invention can be directly used in the treatment of diseases, for example, it can treat various diseases, especially various tumors, embryonic development disorders, growth disorders, diseases, and inflammation. , Immune diseases, certain hereditary, blood diseases, etc.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human L1 factor ORF2 reverse transcription-related protein 15.
  • Agonists increase human L1 factor 0RF2 reverse transcription-related protein 15 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing human L1 factor 0RF2 reverse transcription-related protein 15 can be cultured with labeled human L1 factor 0RF2 reverse transcription-related protein 15 in the presence of drugs. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human L1 factor ORF2 reverse transcription-related protein 15 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonist of human L1 factor 0RF2 reverse transcription-related protein 15 can bind to human L1 factor 0RF2 reverse transcription-related protein 15 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide such that The polypeptide cannot perform biological functions.
  • human L1 factor 0RF2 reverse transcription-related protein I 5 can be added to the bioanalytical assay, and the interaction between human L1 factor 0RF2 reverse transcription-related protein 15 and its receptor can be determined by determining the compound Influence to determine if a compound is an antagonist.
  • Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to human L1 factor ORF2 reverse transcription-related protein 15 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. During screening, 15 molecules of human L1 factor 0RF2 reverse transcription-related protein 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 the human L1 factor ORF2 reverse transcription-related protein 15 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human L1 factor 0RF2 reverse transcription-related protein 15 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant, etc.
  • Techniques for preparing monoclonal antibodies to human L1 factor ORF2 reverse transcription-related protein 15 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human B- Cell hybridoma technology, EBV-hybridoma technology, etc.
  • Antibodies against human L1 factor 0RF2 reverse transcription-related protein 15 can be used in immunohistochemical techniques to detect human L1 factor 0RF2 reverse transcription-related protein 15 in biopsy specimens.
  • Monoclonal antibodies that bind to human L1 factor 0RF2 reverse transcription-related protein 15 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins against a specific bead site in the body.
  • human L1 factor 0RF2 reverse transcription-related protein 15 High affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill the human L1 factor 0RF2 reverse transcription-related protein 15 positive cells.
  • the antibodies in the present invention can be used to treat or prevent diseases related to human L1 factor ORF2 reverse transcription-related protein 15.
  • Administration of appropriate doses of antibodies can stimulate or block human L1 factor 0RF2 reverse transcription-related proteins Production or activity of white 15.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human L1 factor 0RF2 reverse transcription-related protein 15.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human L1 factor 0RF2 reverse transcription-related protein 15 detected in the test can be used to explain the importance of human L1 factor 0RF2 reverse transcription-related protein 15 in various diseases and to diagnose human L1 factor 0RF2 reverse transcription Diseases where related protein 15 plays a role.
  • the polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzyme, and can be analyzed by one-dimensional or two-dimensional or three-dimensional gel electrophoresis, and more preferably by mass spectrometry coding.
  • the polynucleotide of human L1 factor 0RF2 reverse transcription-related protein 15 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 L1 factor 0RF2 reverse transcription-related protein 15.
  • Recombinant gene therapy vectors can be designed to express variant human L1 factor 0RF2 reverse transcription-related protein 15 to inhibit endogenous human L1 factor 0RF2 reverse transcription-related protein 15 activity.
  • a variant human L1 factor 0RF2 reverse transcription-related protein 15 may be a shortened human L1 factor 0RF2 reverse transcription-related protein 15 that lacks a signaling functional domain. Although it can bind to downstream substrates, it lacks Signaling activity. Therefore, the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human L1 factor 0RF2 reverse transcription-related protein 15.
  • 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 the human L1 factor 0RF2 reverse transcription-related protein 15 into cells.
  • viruses such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc.
  • viruses such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding the human L1 factor ORF2 reverse transcription-related protein 15 can be found in the existing literature (Sambrook, et al.).
  • a polynucleotide encoding human L1 factor 0RF2 reverse transcription-related protein 15 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 RM and DM
  • ribozymes that inhibit 15 mRM of human L1 factor ORF2 reverse transcription-related protein are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RM molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense MA, DNA and ribozymes can be obtained by any conventional RNA or MA synthesis technology. For example, solid-phase phosphate amide chemical synthesis to synthesize oligonucleotides 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 DM sequence has been integrated into the Somatic downstream of the 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 L1 factor 0RF2 reverse transcription-related protein 15 can be used for the diagnosis of diseases related to human L1 factor 0RF2 reverse transcription-related protein 15.
  • the polynucleotide encoding human L1 factor 0RF2 reverse transcription-related protein 15 can be used to detect the expression of human L1 factor 0RF2 reverse transcription-related protein 15 or the abnormal expression of human L1 factor 0RF2 reverse transcription-related protein 15 in disease states. . Such as the expression of human L1 factor 0RF2 reverse transcription-related protein 15.
  • Hybridization techniques include Southern blotting, Nor thern blotting, in situ hybridization, and the like. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Human L1 factor 0RF2 reverse transcription-related protein 15 specific primers can be used for MA-polymerase chain reaction (RT-PCR) in vitro amplification to detect the transcription products of human L1 factor 0RF2 reverse transcription-related protein 15.
  • Human L1 factor 0RF2 reverse transcription-related protein 15 gene can also be used to diagnose human L1 factor 0RF2 reverse transcription-related protein 15-related diseases.
  • Human L1 factor 0RF2 reverse transcription-related protein 15 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human L1 factor 0RF2 reverse transcription-related protein 15 DM sequences. Mutations can be detected using existing techniques such as Southern imprinting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, the Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for marking chromosome positions.
  • an important first step is to locate these DM sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared according to cDM, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DM to specific chromosomes.
  • oligonucleotide primers of the present invention 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 and hybrid pre-selection using labeled flow sorting to construct a chromosome-specific CDM library.
  • 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, Mendelian Inheritance in Man (available online with Johns Hopkins University 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 CDM or genomic sequence differences between the affected and the affected individuals need to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable using cDNA sequence-based PCR. Based on 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 a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human L1 factor 0RF2 reverse transcription-related protein 15 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human L1 factor 0RF2 reverse transcription-related protein 15 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine humaine 15 associée à la transcription inverse ORF2 du facteur L1, et un polynucléotide codant ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, des troubles du développement, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant la protéine humaine 15 associée à la transcription inverse ORF2 du facteur L1.
PCT/CN2001/000981 2000-06-19 2001-06-18 Nouveau polypeptide, proteine humaine 15 associee a la transcription inverse orf2 du facteur l1, et polynucleotide codant ce polypeptide WO2002004503A1 (fr)

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Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
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DATABASE GENBANK [online] 26 August 1999 (1999-08-26), "Reverse transcriptase homolog - human transposon L1.1.", retrieved from 1275aa accession no. LOCUS Database accession no. S65824 *
HATTORI M. ET AL.: "L1 family of repetitive DNA sequences in primates may be derived from a sequence encoding a reverse transcriptase-related protein", NATURE, vol. 321, no. 6070, 1986, pages 625 - 628 *
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