WO2002004631A1 - Nouveau polypeptide, proteine 11 humaine associee a la transcription inverse orf2 du facteur l1, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, proteine 11 humaine associee a la transcription inverse orf2 du facteur l1, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002004631A1
WO2002004631A1 PCT/CN2001/001001 CN0101001W WO0204631A1 WO 2002004631 A1 WO2002004631 A1 WO 2002004631A1 CN 0101001 W CN0101001 W CN 0101001W WO 0204631 A1 WO0204631 A1 WO 0204631A1
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polypeptide
human
polynucleotide
factor
reverse transcription
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PCT/CN2001/001001
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English (en)
Chinese (zh)
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Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
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Priority to AU93632/01A priority Critical patent/AU9363201A/en
Publication of WO2002004631A1 publication Critical patent/WO2002004631A1/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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, a human L1 factor, 0RF2 reverse transcription-related protein 11, 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).
  • Members of the L1 family exist in different types of cells, including multifunctional embryonic cells. This proves that the L1 family originates from the same sequence coding protein and is distributed 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 of chromosome lq. Structurally, the L 1 factor does not have long terminal repeats, and its 3, -terminal poly A tail is special, and it is not fixed on each L 1 factor.
  • the L1 factor proliferation process includes transcription, reverse transcription, and reintegration into a new site on the genome.
  • the 5 'end of the L1 factor is mostly a blunt-end structure, which is different from the general sticky-end structure, but does not affect its transcription integration process. 5.
  • the terminal untranslated region (UTR) contains an internal promoter to regulate and control the speed of the entire reverse transcription process.
  • the LRE2 site contains two complete open reading frames (0RF1 and 0RF2), where the 0RF1 frame encodes an approximately 40Kd embryonal cancer cell protein, and the 0RF2 frame is sequenced with reverse transcriptase and other retroviral proteins Similarity can mediate the process of reverse transcription. ⁇ The first 32Bp of L1 factor was removed by experimental means, and the obtained product showed only 20% of normal transcription efficiency.
  • L1 family sequence An open reading frame of the L 1 family sequence is similar to some viral RNA-dependent DM polymerases. It further proves that the L1 family or its ancestors are related to reverse transcriptase. The protein it encodes is actually a Kind of reverse transcriptase.
  • the polypeptide of the present invention is 77% identical to the ORF2 reverse transcriptase region of the L1 factor described above, and has 83% similarity, and contains its characteristic conserved sequences. Therefore, this protein is considered to be a new type L1
  • the factor RTF2-related reverse transcriptase has some biological functions related to reverse transcriptase, and is named human L1 factor RFF2 reverse transcription-related protein 11.
  • the human L1 factor 0RF2 reverse transcription-related protein 11 protein plays an important role in regulating important functions of the body such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes. Therefore, the identification of Many human L1 factors, ORF2, are involved in these processes and are related to the reverse transcription-related protein 1 1 protein, especially the amino acid sequence of this protein.
  • the newcomer's L1 factor, 0RF2 reversal record-related protein 11 protein isolation 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 developing diagnostic and / or therapeutic drugs for the disease, so isolating its coding DNA is important. Disclosure of invention
  • One aspect of the present invention is to provide isolated new polypeptides-human L1 factor 0RF2 reverse transcription-related protein 11 and fragments, analogs and derivatives thereof.
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding the human L1 factor ORF2 reverse transcription-related protein 11.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human L1 factor ORF2 reverse transcription-related protein 1 1.
  • Another object of the present invention is to provide a method for producing human L1 factor ORF2 reverse transcription-related protein 11.
  • Another object of the present invention is to provide antibodies against the polypeptide of the present invention-human L1 factor ORF2 reverse transcription-related protein 11.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention-human L1 factor ORF2 reverse transcription-related protein 11.
  • 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 11.
  • 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:
  • polynucleotide complementary to the polynucleotide ( a );
  • sequence of the polynucleotide is one selected from the group consisting of: (a) having SEQ ID NO: 1
  • 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 the activity of human L1 factor ORF2 reverse transcription related protein 11 protein, 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 11 protein in vitro, which comprises detecting mutations in the polypeptide or a polynucleotide sequence encoding the same 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 11 .
  • 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 fragments or portions 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 may have "conservative" changes in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine.
  • Variants 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 11, 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 can bind to the human L1 factor ORF2 reverse transcription-related protein 11.
  • Antagonist refers to a biological activity or immunological activity that can block or regulate human L1 factor 0RF2 reverse transcription-related protein 11 when combined with human L1 factor 0RF2 reverse transcription-related protein 11.
  • Molecule Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that binds human L1 factor 0RF2 reverse transcription-related protein 11.
  • Regular refers to changes in the function of human L1 factor 0RF2 reverse transcription-related protein 11, including increased or decreased protein activity, changes in binding characteristics, and any other biological properties of human L1 factor 0RF2 reverse transcription-related protein 11. , Functional or immune properties.
  • substantially pure ' means essentially 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 ORF2 reverse transcription-related proteins using standard protein purification techniques. 11. Basically pure human L1 factor 0RF2 reverse transcription-related protein 11 can generate a single main band on a non-reducing polyacrylamide gel. The purity of human L1 factor 0RF2 reverse transcription-related protein 11 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 (Sou thern blotting or Nor thern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean strict Conditions with reduced sex allow non-specific binding because conditions with reduced stringency require that the two sequences bind to each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are the same or similar in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, such as through the MEGALIGN program
  • the MEGALIGN program can compare two or more sequences according to different methods such as the Clus ter method (Hi gg ins, D. G. and P. M. Sharp (1988) Gene 73: 237-244).
  • the Clus ter method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups.
  • the percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: The number of matching residues between sequence A and sequence X 100 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 the Clus ter method or using methods known in the art such as Jotun Hein. The percent identity between nucleic acid sequences (He in J., (1990) Methods in emzumo logy 183: 625- 645).
  • Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitutions for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid link 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,? (& 1) ') 2 and ⁇ , which can specifically bind to the antigenic determinant of human L1 factor ORF2 reverse transcription-related protein 11.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it occurs naturally).
  • a naturally occurring polynucleotide or polypeptide exists in a living animal. It is not isolated, 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 11 refers to human L1 factor 0RF2 reverse transcription-related protein 11 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 0RF2 reverse transcription-related protein 11 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 11 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, a human L1 factor 0RF2 reverse transcription-related protein 11, which basically consists of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be 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 11.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human L1 factor ORF2 reverse transcription related protein 11 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution The amino acid may or may not be encoded by a genetic codon; or (II) a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or ( ⁇ ⁇ ) Such a type, in which the mature polypeptide is fused with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV) such a type, in which the additional amino acid sequence is fused into the mature polypeptide to form a polypeptide sequence (Such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protease sequence) As set forth herein, such fragments, derivatives, and analogs are considered to be within the knowledge of those
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 1916 bases in length and its open reading frame of 1525-1821 encodes 98 amino acids. According to the amino acid sequence homology comparison, it was found that this polypeptide has 77% homology with human L1 factor 0RF2 reverse transcription-related protein. It can be concluded that the human L1 factor 0RF2 reverse transcription-related protein 11 has human L1 factor 0RF2 reverse transcription. Similar structures and functions of related proteins.
  • the polynucleotide of the present invention may be in the DM form or the RM form.
  • DNA forms include cDNA, genomic DM, or synthetic DNA.
  • DNA 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 in the present invention, but which differs from the coding region sequence shown in SEQ ID NO: 1.
  • 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.
  • "strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1 ° /.
  • hybridizable polynucleotide is identical to that shown in SEQ ID NO: 2 Mature peptides have the same biological function and activity.
  • the invention also relates to nucleic acid fragments that hybridize to the sequences described above.
  • nucleic acid fragment contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, 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 ORF2 reverse transcription-related protein 11.
  • 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 11 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 MA sequence to obtain the double-stranded DM 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 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. There are many mature techniques for mRNA extraction. Kits are also commercially available (Q i agene). And the construction of cDNA libraries is also a common method (Sambrook, et al., Mol ecu l ar Cl oni ng, A Labora t ory Manua l, Col d Spr ing Harbor Labora tory. 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 screened from these cDNA libraries by conventional methods. These methods include (but are not limited to): (1) 0 -0 or 0 ⁇ -1 hybridization; (2) the appearance or loss of marker gene function; (3) determination of the transcript of human L1 factor 0RF2 reverse transcription-related protein 11 (4) Detecting the protein product 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 2,000 nucleotides, and preferably within 1,000 nucleotides.
  • the probe used here is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the expression of the human L1 factor 0RF2 reverse transcription-related protein 11 gene is detected.
  • the white product can be obtained by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • the RACE method RACE-cDM terminal rapid amplification method
  • the primers used for PCR may be appropriately based on the polynucleotide sequence information of the present invention disclosed herein.
  • 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 determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, the sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using human L1 factor ORF2 reverse transcription related protein 11 coding sequence, and the recombinant technology to produce the Said method of polypeptide.
  • a polynucleotide sequence encoding the human L1 factor 0RF2 reverse transcription-related protein 11 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 (Rosenberg, et al. Gene, 1987,
  • pMSXND expression vectors (Lee and Nathans, J Bio Chem. 263: 3521, 1988) expressed in mammalian cells and baculovirus-derived vectors expressed in insect cells.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct an expression vector containing a DNA sequence encoding human L1 factor ORF2 reverse transcription-related protein 11 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 DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenoviral enhancers.
  • 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 11 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 s melanoma cells, etc. .
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of DNA uptake can be in the exponential growth phase were harvested, treated with CaC l 2 method used in steps well known in the art. The alternative is to use MgC l 2 .
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human L1 factor 0RF2 reverse transcription-related protein 11 (Scence, 1984; 224: 14 31). 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.
  • recombinant proteins can be separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography
  • FIG. 1 is a comparison diagram of amino acid sequence homology between the inventor's L1 factor 0RF2 reverse transcription-related protein 11 and human L1 factor 0RF2 reverse transcription-related protein.
  • the upper sequence is human L1 factor 0RF2 reverse transcription-related protein 11, 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 11.
  • 1 lkDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using Quik mRNA I solat ion Kit (product of Qiegene). 2ug poly (A) mRM forms cDNA by reverse transcription.
  • a Smart cDNA cloning kit (purchased from Clontech) was used to insert the 00 fragment into a multi-cloning site of a pBSK (+) vector (Clontech) to transform DH5 ⁇ to form a cDNA library.
  • the terminate cycle react ion sequencing kit (Perkin-Elmer) and the ABI 377 automatic sequencer (Perkin-Elmer) determined the sequences at the 5 'and 3' ends of all clones.
  • the determined cDNA sequence was compared with the existing public DM sequence database (Genebank), and it was found that the cDM sequence of one of the clones 1314d09 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • the 1314d09 clone contains a full-length cDNA of 1916bp (as shown in Seq ID NO: l), and has a 297bp open reading frame (0RF) from 1525bp to 1821bp, encoding a new protein (such as Seq ID NO : Shown in 2).
  • This clone pBS-1314d09 and named the encoded protein as human L1 factor 0RF2 reverse transcription-related protein 11.
  • Example 2 Homologous search of cDNA clones
  • the sequence of the human L1 factor 0RF2 reverse transcription-related protein 11 and the protein sequence encoded by the invention are:
  • Blas t program (Basiclocal Al ignment search tool) [Al tschul, SF et a l.
  • CDNA was synthesized using fetal brain cells as a template and ol igo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, the following primers were used for PCR amplification:
  • Pr imerl 5'- AGGCAGTAATAGTCTACCAAAAAA-3, (SEQ ID NO: 3)
  • Primer 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 conditions 50 ⁇ l of KC1, 10mraol / L Tri s-
  • CI (pH 8. 5), 1.5 crypto ol / L MgCl 2 , 200 ⁇ mol / L dNTP, 1 Opmol primer, 1 U Taq DNA polymerase (C 1 on Tech company product).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perk in-Elmer) for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2rain 0 ⁇ -act in For positive control and template blank as negative control.
  • the amplified product was purified using a QIAGEN kit and ligated to a PCR vector using a TA cloning kit (Invitrogen).
  • Example 4 Analysis of human LI factor 0RF2 reverse transcription-related protein 11 gene expression by Northern blot method: Total RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] 0
  • This method includes acid guanidinium thiocyanate -Chloroform extraction. That is, the tissue is homogenized with 4M guanidinium isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ), Mix and centrifuge.
  • RNA precipitate Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water. Using 20 g of RNA, electrophoresis was performed on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 mM EDTA-2.2M formaldehyde. It was then transferred to a nitrocellulose membrane. Preparation 32 P- DNA probe labeled with a- 32 P dATP by random priming method.
  • the DNA probe used was the PCR-encoded human L1 factor ORF2 reverse transcription-related protein 11 coding region sequence (1525bp to 1821bp) 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-25mM KH 2 P0 4 (pH7.4)-5xSSC-5x Denhardt's solution and 200 g / ml salmon sperm DNA. After hybridization, the filter was washed in 1 x SSC-0.1% SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 5 In vitro expression, isolation and purification of recombinant human L1 factor 0RF2 reverse transcription-related protein 11
  • Primer3 5'-CATCCATGGATGAAACCCAAAACTATAAAAACC-3 '(Seq ID No: 5)
  • Primer4 5'-CATGGATCCCCATGGTTTTTTTTCTCGTAAAT-3' (Seq ID No: 6)
  • the 5 'ends of these two primers contain Ncol and BamHI restriction sites, respectively.
  • the following are the coding sequences of the 5 'and 3' ends of the gene of interest, respectively.
  • the Ncol and BamHI restriction sites correspond to the selectivity on the expression vector plasmid pET- 2 8b (+) (Novagen, Cat. No. 69865.3). Endonuclease site.
  • the PCR reaction was performed using pBS-1314d09 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-131 4 d0 9 plasmid, primers Primer-3 and Primer- 4 points, and 1 J was 10 pmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1.
  • 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 the coliform bacteria DH5 ⁇ by the calcium chloride method, and cultured overnight in LB plates containing kanamycin (final concentration 30 ⁇ ⁇ / ⁇ 1), and then positive clones were selected by colony PCR method and sequenced.
  • a positive clone (pET-1314d09) 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.
  • the host strain BL21 (pET-1314d09) was 37 in LB liquid medium containing kanamycin (final concentration 30 ⁇ g / ml).
  • NH2-Met-Lys-Pro-Lys-Thr-I le-Lys-Thr-Pro-Glu-Glu-Asn-Leu-Gly-Asp- C00H (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 the method, see: Avraraeas, et al. Immunochemi s try, 1969; 6: 43. Rabbits were immunized with 4rag above-mentioned cyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost the immunity once.
  • 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 serum.
  • 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 11.
  • 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 a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by 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 saturated with the carrier and the synthetic polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes, and Incubation hybridizes the probe to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, 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:
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membrane nitrocellulose membrane
  • the 32 P-Probe (the second peak is free ⁇ - 32 P-dATP) is prepared.
  • polypeptides of the present invention can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases. ⁇
  • 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 cDNA 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 the reverse transcriptase and other retroviral proteins Sequence similarity can mediate the reverse transcription process.
  • the protein encoded by the L1 family sequence is actually a reverse transcriptase-like enzyme.
  • the polypeptide of the present invention is highly homologous with the human RFL2 type RFF2 reverse transcriptase, and contains the characteristic sequences of the RF1 type RFF2 reverse transcriptase family, 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 metabolic disorders, protein metabolic disorders, and related tissue tumors and cancers, and produce related diseases. It can be seen that the abnormal expression of the human LI factor 0RF2 reverse transcription-related protein 1 1 of the present invention will produce various diseases, especially various tumors, embryonic development disorders, growth disorders, inflammation, and immune diseases. Illnesses 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 retardation, brain development disorders, skin, fat, and muscular dysplasia, bone and joint dysplasia, various metabolic defects, 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 ulcer, cervicitis, Various infectious inflammations
  • Immune diseases Systemic lupus erythematosus, rheumatoid arthritis, bronchial asthma, urticaria, specific dermatitis, post-infection myocarditis, scleroderma, myasthenia gravis, Guillain-Barre syndrome, common variable immunodeficiency disease , Primary B-lymphocyte immunodeficiency disease, Acquired immunodeficiency syndrome
  • the abnormal expression of the human L1 factor 0RF2 reverse transcription-related protein 11 of the present invention will also generate certain hereditary, hematological diseases and the like.
  • polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially various tumors, embryonic development disorders, growth and development disorders, inflammation, and immunity Sexual 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 0RF2 reverse transcription-related protein 11.
  • Agonists enhance human L1 factor 0RF2 reverse transcription-related protein 11 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • a mammalian cell or a membrane preparation expressing human L1 factor 0RF2 reverse transcription-related protein 11 and a labeled human L1 factor 0RF2 reverse transcription-related protein can be used.
  • Antagonists of human L1 factor 0RF2 reverse transcription-related protein 11 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonist of human L1 factor 0RF2 reverse transcription-related protein 11 can bind to human L1 factor 0RF2 reverse transcription-related protein 11 and eliminate its function, or inhibit the production of the polypeptide, Or binding to the active site of the polypeptide prevents the polypeptide from performing a biological function.
  • human L1 factor 0RF2 reverse transcription-related protein 11 can be added to the bioanalytical assay, and the interaction between human L1 factor 0RF2 reverse transcription-related protein 11 and its receptor can be determined by determining the compound Influence to determine if a compound is an antagonist.
  • antagonist deletions and analogs can be screened. Can interact with human L1 factor 0RF2
  • Peptide molecules bound by reverse transcription-related protein 11 can be obtained by screening a random peptide library consisting of various possible combinations of amino acids bound to a solid phase. In the screening, 11 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 11 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 11 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 0RF2 reverse transcription-related protein 11 include, but are not limited to, hybridoma technology (Kohl er and i l s t e in. Nature, 1975,
  • Antibodies against human L1 factor 0RF2 reverse transcription-related protein 11 can be used in immunohistochemical techniques to detect human L1 factor 0RF2 reverse transcription-related protein 11 in biopsy specimens.
  • Monoclonal antibodies that bind to human L1 factor 0RF2 reverse transcription-related protein 11 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • human L1 factor 0RF2 reverse transcription-related protein 11 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 human L1 factor 0RF2 reverse transcription-related proteins 11 positive cells.
  • the antibody of the present invention can be used to treat or prevent human L1 factor ORF2 reverse transcription related protein 11 phase Related diseases.
  • Administration of an appropriate dose of antibody can stimulate or block the production or activity of human LI factor ORF2 reverse transcription-related protein 11.
  • the present invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human L1 factor 0RF2 reverse transcription-related protein 11.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human L1 factor 0RF2 reverse transcription-related protein 11 detected in the test can be used to explain the importance of human L1 factor 0RF2 reverse transcription-related protein 11 in various diseases and to diagnose human L1 factor 0RF2 reverse transcription Diseases in which related protein 11 plays a role.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • the polynucleotide encoding human L1 factor 0RF2 reverse transcription-related protein 11 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 11.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutant human L1 factor 0RF2 reverse transcription-related protein 11 to inhibit endogenous human L1 factor 0RF2 reverse transcription-related protein 11 activity.
  • a variant human L1 factor 0RF2 reverse transcription-related protein 11 may be shortened and lack the signaling functional domain of human L1 factor 0RF2 reverse transcription-related protein 11.
  • the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of human L1 factor 0RF2 reverse transcription-related protein 11.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding human L1 factor 0RF2 reverse transcription-related protein 11 into cells.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding the human L1 factor 0RF2 reverse transcription-related protein 11 can be found in the literature (Sambrook, et al.).
  • a polynucleotide encoding human L1 factor 0RF2 reverse transcription-related protein 11 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 L1 factor ORF2 reverse transcription-related protein 11 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that can specifically decompose a specific RM. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA and performs endonucleation.
  • Antisense RNA and DNA and ribozymes can be obtained by any RNA or DNA synthesis technology, such as the technology for the synthesis of oligonucleotides by solid-phase phosphate amide chemical synthesis, which is widely used.
  • Antisense RNA molecules can Obtained by transcription of a DNA sequence encoding the RNA 'in vitro or in vivo. This DNA sequence has been integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of a nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside linkages should use phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human L1 factor 0RF2 reverse transcription-related protein 1 1 can be used for diagnosis of diseases related to human L1 factor 0RF2 reverse transcription-related protein 11.
  • the polynucleotide encoding human L1 factor 0RF2 reverse transcription-related protein 11 can be used to detect the expression of human L1 factor 0RF2 reverse transcription-related protein 11 or the abnormal expression of human L1 factor 0RF2 reverse transcription-related protein 11 in disease states.
  • the DNA sequence encoding human L1 factor 0RF2 reverse transcription-related protein 11 can be used to hybridize biopsy specimens to determine the expression status of human L1 factor 0RF2 reverse transcription-related protein 11.
  • Hybridization techniques include Southern blotting, Nor thern imprinting, and in situ hybridization. These techniques and methods are all mature and open technologies, and related kits are commercially available. Some or all of the polynucleotides of the present invention can be used as probes to be fixed on a micro-array (Mi croar ray) or a DNA chip (also known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues. .
  • Human L1 factor 0RF2 reverse transcription-related protein 1'1 specific primers can also be used to detect the transcription products of human L1 factor 0RF2 reverse transcription-related protein 11 by MA-polymerase chain reaction (RT-PCR) in vitro amplification.
  • Human L1 factor 0RF2 reverse transcription-related protein 1 1 gene mutations can also be used to diagnose human L1 factor 0RF2 reverse transcription-related protein 11-related diseases.
  • Human L1 factor 0RF2 reverse transcription-related protein 11 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type human L1 factor 0RF2 reverse transcription-related protein 11 DNA sequences. Mutations can be detected using existing techniques such as Southern imprinting, MA 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 DNA sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared from the cDNA, and the sequences can be located on the chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention by a similar method, a set of fragments from a specific chromosome can be utilized Or a large number of genomic clones 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
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. Based on the resolution capabilities of current physical mapping and gene mapping technologies, the cDM that is accurately mapped to a disease-related chromosomal region can be one of 50 to 500 potentially pathogenic genes (assuming
  • 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 L1 factor 0RF2 reverse transcription-related protein 11 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 11 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine 11 humaine 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 11 humaine associée à la transcription inverse ORF2 du facteur L1.
PCT/CN2001/001001 2000-06-19 2001-06-18 Nouveau polypeptide, proteine 11 humaine associee a la transcription inverse orf2 du facteur l1, et polynucleotide codant ce polypeptide WO2002004631A1 (fr)

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CN 00116557 CN1329028A (zh) 2000-06-19 2000-06-19 一种新的多肽——人l1因子orf2反转录相关蛋白11和编码这种多肽的多核苷酸
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Non-Patent Citations (5)

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Title
DATABASE GENBANK [online] 16 January 1999 (1999-01-16), Database accession no. AC006213 *
DATABASE GENBANK [online] 17 December 1999 (1999-12-17), Database accession no. AC018725 *
DATABASE GENBANK [online] 21 December 1999 (1999-12-21), Database accession no. AC004453 *
DATABASE GENBANK [online] 21 May 1999 (1999-05-21), Database accession no. AC007262 *
DATABASE GENBANK [online] 6 April 2000 (2000-04-06), Database accession no. AC035150 *

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