A novel polypeptide, a human reverse transcription relative factor 33 and the polynucleotide encoding the polypeptide

Abstract

The present invention discloses a novel polypeptide, a human reverse transcription relative factor 33, the polynucleotide encoding the polypeptide and the method for producing the polypeptide by DNA recombinant technology. The invention also discloses the uses of the polypeptide in methods for treating various diseases, such as dysmyotrophy, malignant tumour, hemopathy, HIV infection, immunological disease, and various inflammations, etc. The invention also discloses the agonists against the polypeptide and the therapeutic action thereof. The invention also discloses the uses of the polynucleotide encoding the novel human reverse transcription relative factor 33.

Description

[0001] A novel polypeptide—human reverse transcription-associated factor 33 and the polynucleotide encoding this polypeptide—are in the field of technology.

[0002] This invention belongs to the field of biotechnology. Specifically, this invention describes a novel polypeptide—human reverse transcription-associated factor 33—and the polynucleotide sequence encoding this polypeptide. This invention also relates to methods for preparing this polynucleotide and polypeptide, and their applications.

[0003] Background Technology

[0004] L1 factors can be found in all mammalian genomes and participate in reverse transcription. In the human genome, it is located on LRE2 of chromosome lq. Structurally, L1 factors do not have long terminal repeat sequences; their 3'-terminal poly-A tail is unique and not fixed on every L1 factor. The proliferation of L1 factors involves transcription, reverse transcription, and reintegration into a new site on the genome. The 5' ends of L1 factors are mostly blunt-ended, unlike typical sticky ends, but this does not affect their transcriptional integration. The 5' untranslated region (UTR) contains an intrinsic promoter that regulates and controls the rate of the entire reverse transcription process. The LRE2 site contains two complete open reading frames (ORF1 and ORF2). ORF1 encodes an approximately 40 kDa embryonic cancer cell protein, while ORF2 shares sequence similarity with reverse transcriptase and other reverse transcriptase viral proteins and mediates the reverse transcription process. Removing the first 32Bp of the L1 factor using experimental methods yielded a product that only showed 20% of the normal transcription efficiency.

[0005] The insertion at LRE2 demonstrates that the flanking sequences generated by the L1 factor during readthrough can undergo reverse transcription, due to the presence of different polyA structures at its tail. In fact, this type of readthrough can also be achieved via retroviruses, similarly yielding the desired intracellular DNA sequence. Insertion rearrangements at the LRE2 site can lead to some abnormalities in gene expression. A typical example is muscular dystrophy. In the muscles of malnourished patients, a dystrophy protein has been found with an approximately 2Kb insertion gene containing a rearranged L1 factor. The patient's chromosome lq has an allele at the LRE2 site, corresponding to a complete 13-15B LRE2 sequence. PThe target site copy also has an unusual 21-bp blunt end at the 5' end, demonstrating that the blunt-end structure can still perform reverse transcription. Furthermore, the insertion of two bases on the L1 factor leads to Duchenne muscular dystrophy (B, Bakker, & G. van Omenn, persona 1 communication), a familial disease in which individuals typically die in childhood or early adulthood. The polypeptide of this invention shares 90% identity and 94% similarity with the aforementioned L1 factor and contains L1. Because of the conserved sequence characteristic of the family, this protein is considered to be a new L1 factor with similar biological functions to the L1 factor, and has been named human reverse transcription-associated factor 33.

[0006] As mentioned above, human reverse transcription factor 33 (RTF-33) plays a crucial role in regulating vital bodily functions such as cell division and embryonic development. Given that these regulatory processes are believed to involve numerous proteins, there has been a long-standing need in the art to identify more RTF-33 proteins involved in these processes, particularly to determine their amino acid sequences. The isolation of the gene encoding the novel human RTF-33 protein provides a foundation for determining its role in healthy and disease states. This protein may form the basis for developing diagnostic and / or therapeutic agents for diseases; therefore, isolating its encoding DNA is of paramount importance. Disclosure of the Invention

[0007] One object of the present invention is to provide a novel isolated polypeptide—human reverse transcription-associated factor 33, as well as its fragments, analogs and derivatives.

[0008] Another object of the present invention is to provide a polynucleotide encoding the polypeptide.

[0009] Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding human reverse transcription-associated factor 33.

[0010] Another object of the present invention is to provide genetically engineered host cells containing a polynucleotide encoding human reverse transcription-associated factor 33.

[0011] Another object of the present invention is to provide a method for producing human reverse transcription-related factor 33.

[0012] Another object of the present invention is to provide an antibody against the polypeptide of the present invention—human reverse transcription-associated factor 3.

[0013] Another object of the present invention is to provide mimicry compounds, antagonists, agonists, and inhibitors of the polypeptide of the present invention—human reverse transcription-associated factor 3.

[0014] Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities in human reverse transcription factor 33.

[0015] This invention relates to an isolated polypeptide, which is of human origin, comprising: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conserved variant thereof, a bioactive fragment, or a derivative thereof. Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0016] The present invention also relates to an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:

[0017] (a) A polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID No. 2;

[0018] (b) A polynucleotide complementary to polynucleotide (a);

[0019] (c) A polynucleotide that has at least 91% identity with the polynucleotide sequence of (a) or (b). More preferably, the sequence of the polynucleotide is selected from one of the following groups: (a) a sequence having positions 1426-2328 of SEQ ID NO: 1; and (b) a sequence having positions 1-2707 of SEQ ID NO: 1.

[0020] The present invention also relates to a vector containing the polynucleotides of the present invention, particularly an expression vector; a host cell genetically engineered using the vector, including a transformed, transduced, or transfected host cell; and a method for preparing the polypeptides of the present invention, including culturing the host cell and recovering the expression product.

[0021] The present invention also relates to an antibody that can specifically bind to the polypeptide of the present invention.

[0022] The present invention also relates to a method for screening compounds that mimic, activate, antagonize, or inhibit the activity of human reverse transcription-associated factor 33 protein, comprising a polypeptide utilizing the present invention. The present invention also relates to compounds obtained using this method.

[0023] The present invention also relates to a method for in vitro detection of diseases or disease susceptibility associated with abnormal expression of human reverse transcription-associated factor 33 protein, comprising detecting mutations in the polypeptide or its encoding polynucleotide sequence in a biological sample, or detecting the amount or biological activity of the polypeptide of the present invention in a biological sample.

[0024] The present invention also relates to a pharmaceutical composition comprising the polypeptide of the present invention or an analogue thereof, an activator, an antagonist or inhibitor, and a pharmaceutically acceptable carrier.

[0025] The present invention also relates to the use of the polypeptides and / or polynucleotides of the present invention in the preparation of medicaments for treating cancer, developmental disorders or immune disorders or other diseases caused by abnormal expression of human reverse transcription-associated factor 33.

[0026] Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein. Unless otherwise specified, the following terms as used in this specification and claims have the following meanings: "nucleic acid sequence" refers to oligonucleotides, nucleotides, or polynucleotides and fragments or portions thereof, and may also refer to genomic or synthetic DNA or RNA, which may be single-stranded or double-stranded, representing sense or antisense strands. Similarly, the term "amino acid sequence" refers to oligopeptides, peptides, polypeptides, or protein sequences and fragments or portions thereof. When "amino acid sequence" in this invention refers to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide" or "protein" does not mean limiting the amino acid sequence to the complete natural amino acids associated with said protein molecule.

[0027] A protein or polynucleotide "variant" is an amino acid sequence or polynucleotide sequence encoding it that has one or more amino acid or nucleotide changes. These changes may include the deletion, insertion, or substitution of amino acids or nucleotides in the amino acid or nucleotide sequence. Variants may have "conserved" changes, where the substituted amino acid has a similar structure or chemical properties to the original amino acid, such as replacing isoleucine with leucine. Variants may also have non-conserved changes, such as replacing glycine with tryptophan. "Deficiency" refers to the absence of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.

[0028] "Insertion" or "addition" refers to a change in the amino acid or nucleotide sequence that results in the addition of one or more amino acids or nucleotides compared to the naturally occurring molecule. "Replacement" refers to the substitution of one or more amino acids or nucleotides by different amino acids or nucleotides.

[0029] "Bioactivity" refers to proteins that possess the structural, regulatory, or biochemical functions of natural molecules. Similarly, the term "immunologic activity" refers to the ability of natural, recombinant, or synthetic proteins and fragments thereof to induce specific immune responses and bind to specific antibodies in suitable animals or cells.

[0030] An "agonist" is a molecule that, when bound to human reverse transcription factor 33, causes alterations in the protein, thereby regulating its activity. Agonists can include proteins, nucleic acids, carbohydrates, or any other molecules that can bind to human reverse transcription factor 33.

[0031] "Antagonist" or "inhibitor" is a molecule that, when bound to human reverse transcription factor 33, blocks or modulates the biological or immunological activity of human reverse transcription factor 33. Antagonists and inhibitors can include proteins, nucleic acids, carbohydrates, or any other molecules that can bind to human reverse transcription factor 33.

[0032] "Regulation" refers to changes in the function of human reverse transcription-associated factor 33, including increases or decreases in protein activity, changes in binding properties, and any other changes in the biological, functional, or immune properties of human reverse transcription-associated factor 33.

[0033] "Very pure" means that it is substantially free of other naturally occurring or related proteins, lipids, carbohydrates, or other substances. Those skilled in the art can purify human reverse transcription factor 33 using standard protein purification techniques. Virtually pure human reverse transcription factor 33 produces a single master band on a non-reducing polyacrylamide gel. The purity of the human reverse transcription factor 33 peptide can be determined by amino acid sequence analysis.

[0034] "Complementary" or "complementary" refers to the natural binding of polynucleotides through base pairing under permissible salt concentration and temperature conditions. For example, the sequence "C-T-G-A" can bind to the complementary sequence "G-A-C-T". Complementarity between two single-stranded molecules can be partial or complete. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands.

[0035] "Homology" refers to the degree of complementarity, which can be partial or complete homology. "Partial homology" refers to a partially complementary sequence that at least partially inhibits the hybridization of a completely complementary sequence with the target nucleic acid. This inhibition of hybridization can be detected by hybridization under reduced stringency conditions (such as Soutern blotting or Northern blotting). Essentially, homologous sequences or hybridization probes can compete for and inhibit the binding of completely homologous sequences to the target sequence under reduced stringency conditions. This does not mean that reduced stringency conditions allow non-specific binding, because reduced stringency conditions require the two sequences to bind to each other. They can interact in a specific or selective manner.

[0036] "Identity percentage" refers to the percentage of identical or similar sequences in a comparison of two or more amino acid or nucleic acid sequences. The identity percentage can be determined electronically, such as using the MEGALIGN program (Lasergene software package, DNASTAR, Inc., Madison Wis.). The MEGALIGN program can compare two or more sequences using different methods, such as the Cluster method (Higgins, DG, and PM Sharp (1988) Gene 73: 237-244). The Cluster method arranges sequences into clusters by examining the distance between all pairs. The clusters are then assigned in pairs or groups. The identity percentage between two amino acid sequences, such as sequence A and sequence B, is calculated using the following formula:

[0037] Number of matching residues between sequence A and sequence B × 100 (Number of residues in sequence A - Number of spacer residues in sequence A - Number of spacer residues in sequence B)

[0038] The percentage of similarity between nucleic acid sequences can also be determined by the cluster method or by methods known in the field, such as Jotun Hein (Hein J., (1990) Methods in emzumology 183: 625-645).

[0039] "Similarity" refers to the degree of similarity or conserved substitution of amino acid residues at corresponding positions when comparing the arrangement of amino acid sequences. Examples of amino acids used for conserved substitution include negatively charged amino acids such as aspartic acid and glutamic acid; positively charged amino acids such as lysine and arginine; amino acids with similar hydrophilicity and uncharged head groups such as leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.

[0040] "Antense" refers to a nucleotide sequence that is complementary to a specific DNA or RM sequence. "Antense strand" refers to a nucleic acid strand that is complementary to the "sense strand".

[0041] "Derivatives" refer to chemical modifications of HFPs or the nucleic acids that encode them. These modifications can involve replacing hydrogen atoms with alkyl, acyl, or amino groups. Nucleic acid derivatives can encode polypeptides that retain the main biological characteristics of the natural molecule.

[0042] "Antibody" refers to the complete antibody molecule and its fragments, such as Fa, ? (^')2 and ?, which can specifically bind to the antigenic determinant of human reverse transcription-associated factor 33.

[0043] "Humanized antibody" refers to an antibody whose non-antigen binding region amino acid sequence has been replaced to make it more similar to a human antibody, but still retains its original binding activity.

[0044] The term "separated" refers to the removal of a substance from its original environment (e.g., its natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide existing in a living organism is not separated, but the same polynucleotide or polypeptide being separated from some or all of the substances that coexist with it in the natural system is considered separated. Such a polynucleotide may be part of a carrier, or it may be part of a composition. Since the carrier or composition is not its natural environment... The components are still isolated. As used in this invention, "isolated" means that the substance has been separated from its original environment (or, if it is a natural substance, the original environment is the natural environment). For example, polynucleotides and polypeptides in their natural state within living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances present in their natural state.

[0045] As used herein, "isolated human reverse transcription-associated factor 33" means that human reverse transcription-associated factor 33 is substantially free of other naturally occurring proteins, lipids, carbohydrates, or other substances associated with it. Those skilled in the art can purify human reverse transcription-associated factor 33 using standard protein purification techniques. A substantially pure peptide will produce a single master band on a non-reducing polyacrylamide gel. The purity of the human reverse transcription-associated factor 33 peptide can be determined by amino acid sequence analysis.

[0046] This invention provides a novel polypeptide—human reverse transcription-associated factor 33—which is essentially composed of the amino acid sequence shown in SEQ ID NO: 2. The polypeptide of this invention can be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, preferably a recombinant polypeptide. The polypeptide of this invention can be a naturally purified product, a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant technology. Depending on the host used in the recombinant production protocol, the polypeptide of this invention can be glycosylated or non-glycosylated. The polypeptide of this invention may or may not include an initial methionine residue.

[0047] This invention also includes fragments, derivatives, and analogs of human reverse transcription-associated factor 33. As used herein, the terms "fragment," "derivative," and "analyte" refer to polypeptides that substantially retain the same biological function or activity as human reverse transcription-associated factor 33 of this invention. Fragments, derivatives, or analogs of the polypeptides of this invention may be: (I) in which one or more amino acid residues are substituted with conserved or non-conserved amino acid residues (preferably conserved amino acid residues), and the substituted amino acids may or may not be encoded by a genetic codon; or (II) in which a group on one or more amino acid residues is substituted with another group containing a substituent; or (II) in which a mature polypeptide is fused with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) in which an additional amino acid sequence is fused into the mature polypeptide to form a polypeptide sequence (e.g., a leader sequence, a secretory sequence, or a sequence used to purify the polypeptide or a proteogen sequence). By way of the description herein, such fragments, derivatives, and analogs are considered to be within the knowledge of those skilled in the art.

[0048] This invention provides isolated nucleic acids (polynucleotides) that are essentially composed of polynucleotides encoding polypeptides having the amino acid sequence of SEQ ID NO: 2. The polynucleotide sequence of this invention includes the nucleotide sequence of SEQ ID NO: 1. The polynucleotide of this invention was discovered from a cDNA library of human fetal brain tissue. It contains a full-length polynucleotide sequence of 2707 bases, with open reading frames 1426-2328 encoding 300 amino acids. Based on amino acid sequence homology comparison, this polypeptide has 90% homology with the L1 factor, suggesting that this human reverse transcription-related factor 33 has a similar structure and function to the L1 factor.

[0049] The polynucleotides of this invention can be in DNA or RNA form. DM form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be identical to the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant. As used in this invention, "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2, but differing from the coding region sequence shown in SEQ ID NO: 1.

[0050] 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) and non-coding sequences.

[0051] The term "polynucleotide encoding a polypeptide" refers to a polynucleotide that includes the polynucleotide encoding the polypeptide and a polynucleotide that includes additional coding and / or non-coding sequences.

[0052] This invention also relates to variants of the polynucleotides described above, which encode polypeptides or polypeptide fragments, analogs, and derivatives having the same amino acid sequence as those of this invention. These polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide it encodes.

[0053] The present invention also relates to polynucleotides that hybridize with the sequences described above (the two sequences having at least...)

[0054] 50%, preferably 70% similarity). This invention particularly relates to polynucleotides that are hybridizable with the polynucleotides described herein under stringent conditions. In this invention, "stringent conditions" means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2xSSC, 0.1... e / . SDS, 60°C; or (2) a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal serum / 0.1% Ficol, 42°C, etc., is added during hybridization; or (3) hybridization occurs only when the similarity between the two sequences is at least 95%, preferably more than 97%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.

[0055] This invention also relates to nucleic acid fragments that hybridize with the sequences described above. As used in this invention, the length of a "nucleic acid fragment" is at least 10 nucleotides, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nucleotides or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding human reverse transcription-associated factor 33.

[0056] The polypeptides and polynucleotides in this invention are preferably provided in isolated form and are more preferably purified to homogenization. The specific polynucleotide sequence encoding human reverse transcription-associated factor 33 of the present invention can be obtained by a variety of methods. For example, the polynucleotide can be isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridizing probes with genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect polynucleotide fragments of clones with common structural features.

[0057] The DM fragment sequence of the present invention can also be obtained by the following methods: 1) isolating double-stranded DNA sequences from genomic DNA; 2) chemically synthesizing DM sequences to obtain double-stranded DNA of the polypeptide.

[0058] Of the methods mentioned above, isolating genomic DNA is the least common. Direct chemical synthesis of DNA sequences is the frequently chosen method. An even more common method is the isolation of cDNA sequences. The standard method for isolating cDNA of interest is to isolate mRNA from donor cells that highly express the gene and perform reverse transcription to form plasmid or phage cDNA libraries. Several mature techniques exist for mRNA extraction, and kits are commercially available (Qiagene). Constructing cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989). Commercially available cDNA libraries are also available, such as various cDNA libraries from Clontech. When combined with polymerase chain reaction technology, even very small amounts of expression products can be cloned.

[0059] The genes of this invention can be screened from these cDNA libraries using conventional methods. These methods include (but are not limited to): (1) DNA-DNA or DM-RNA hybridization; (2) marking the presence or loss of gene function; (3) measuring the level of transcripts of human reverse transcription factor 33; and (4) detecting the protein products of gene expression by immunological techniques or by measuring biological activity. The above methods can be used alone or in combination.

[0060] In method (1), 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, and most preferably at least 100 nucleotides. Furthermore, the length of the probe is generally within 2000 nucleotides, preferably within 1000 nucleotides. The probe used herein is typically a chemically synthesized DNA sequence based on the gene sequence information of the present invention. The gene itself or a fragment of the present invention can, of course, be used as a probe. The DNA probe can be labeled with radioactive isotopes, fluorescein, or enzymes (such as alkaline phosphatase), etc.

[0061] In the fourth method, the protein products of human reverse transcription-related factor 33 gene expression can be detected using immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).

[0062] The method of amplifying DM / RNA using PCR technology (Saiki, et al. Science 1985; 230: 1350-1354) is preferred for obtaining the gene of the present invention. Especially when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE-cDNA end amplification method) is preferred. The primers used for PCR can be appropriately selected based on the polynucleotide sequence information of the present invention disclosed herein and can be synthesized using conventional methods. The amplified MA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0063] The gene of the present invention obtained as described above, or the polynucleotide sequence of various DNA fragments, etc., can be used in common... Standard methods, such as the dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467), are used for determination. These types of polynucleotide sequences can also be determined using commercial sequencing kits. To obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes, it is necessary to determine the cDNA sequences of multiple clones before they can be assembled into a full-length cDNA sequence.

[0064] The present invention also relates to vectors containing the polynucleotides of the present invention, host cells generated by genetic engineering using the vectors of the present invention or directly using the coding sequence of human reverse transcription factor 33, and methods for generating the polypeptides of the present invention via recombinant technology.

[0065] In this invention, a polynucleotide sequence encoding human reverse transcription-associated factor 33 can be inserted into a vector to construct a recombinant vector containing the polynucleotides described in this invention. The term "vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well-known in the art. Vectors applicable in this invention include, but are not limited to: expression vectors based on the T7 promoter expressed in bacteria (Rosenberg, et al. Gene, 1987, 56: 125); pMSXND expression vectors expressed in mammalian cells (Lee and Nathans, J Bio Chem. 263: 3521, 1988); and vectors derived from baculoviruses expressed in insect cells. In short, any plasmid and vector can be used to construct a recombinant expression vector, provided it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain a replication origin, a promoter, a marker gene, and translational regulatory elements.

[0066] Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human reverse transcription-associated factor 33 and suitable transcription / translation regulatory elements. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989). The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include: the lac or trp promoter of *E. coli*; the PL promoter of *λ* phage; eukaryotic promoters including the immediate early promoter of CMV, the thymidine kinase promoter of HSV, early and late SV40 promoters, LTRs of retroviruses, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Inserting enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors of DNA expression, typically consisting of approximately 10 to 300 base pairs, that act on promoters to enhance gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs) located late on the replication origin side, the polyoma enhancer located late on the replication origin side, and adenovirus enhancers.

[0067] Furthermore, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, etc. And green fluorescent protein (GFP), or for tetracycline or ampicillin resistance in E. coli, etc. Those skilled in the art are well aware of how to select appropriate vectors / transcriptional regulatory elements (such as promoters, enhancers, etc.) and selectively mark genes.

[0068] In this invention, a polynucleotide encoding human reverse transcription-associated factor 33 or a recombinant vector containing such polynucleotide can be transformed or introduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector. The term "host cell" refers to prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: *Escherichia coli*, *Streptomyces*; bacterial cells such as *Salmonella typhimurium*; fungal cells such as yeast; plant cells; insect cells such as *Drosophila S2* or *Sf9*; and animal cells such as CHO, COS, or Bowes melanoma cells.

[0069] Transformation of host cells using the DM sequence or a recombinant vector containing the DNA sequence described in this invention can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *Escherichia coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps of which are well known in the art. MgCl2 is also an option. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DM transfection methods can be used: calcium phosphate co-precipitation, or conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0070] Using conventional recombinant DNA technology, the polynucleotide sequence of this invention can be used to express or produce recombinant human reverse transcription-related factor 33 (Science, 1984; 224: 1431). Generally, the following steps are involved:

[0071] (1) Transform or transduce suitable host cells using the polynucleotide (or variant) encoding human reverse transcription-associated factor 33 of the present invention, or using a recombinant expression vector containing the polynucleotide;

[0072] (2) Culture the host cells in a suitable culture medium;

[0073] (3) Isolate and purify proteins from culture media or cells.

[0074] In step (2), the culture medium used in the culture can be selected from various conventional culture media, depending on the host cells used. The cells are cultured under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature change or chemical induction), and the cells are cultured for a further period.

[0075] In step (3), the recombinant polypeptide may be encapsulated within cells, expressed on the cell membrane, or secreted extracellularly. If desired, the recombinant protein may be separated and purified using various separation methods based on its physical, chemical, and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional refolding treatment, protein precipitation (salting out), centrifugation, percolation, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations thereof. Brief description of the attached figures

[0076] The following figures are used to illustrate specific embodiments of the present invention, and are not intended to limit the scope of the invention as defined by the claims.

[0077] Figure 1 is a comparison of the amino acid sequence homology of human reverse transcription-related factor 33 and L1 factor. The upper sequence is human reverse transcription-related factor 33, and the lower sequence is L1 factor. Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+".

[0078] Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) image of isolated human reverse transcription-related factor 33.

[0079] 33 kDa is the molecular weight of the protein. The arrow points to the isolated protein band. The best mode for carrying out this invention.

[0080] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. The experimental methods described in the following embodiments under specific conditions are generally performed according to conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0081] Example 1: Cloning of human reverse transcription-related factor 33

[0082] Total RNA was extracted from human fetal brain using a one-step method of guanidine isothiocyanate / phenol / chloroform. Oa(A) mRNA was isolated from total 1a bacteria using the Quik mRNA Isolation Kit (Clontech). 2 μg of poly(A) mRNA was reverse transcribed to form cDNA. The cDNA fragment was directionally inserted into the multiple cloning site of a pBSK(+) vector (Clontech) using the Smart cDNA Cloning Kit (Clontech), and the bacteria were transformed into DH5oc to form a cDNA library. The 5' and 3' ends of all clones were sequenced using the Dye terminate cycle reaction sequencing kit (PerkinElmer) and an ABI 377 automated sequencer (PerkinElmer). The determined cDNA sequences were compared with existing public DNA sequence databases (Genebank), and the cDNA sequence of clone 0102f02 was found to be novel. A series of primers were synthesized to perform bidirectional sequencing of the inserted cDNA fragment contained in this clone. The results showed that the full-length cDNA contained in clone 0102f02 was 2707 bp (e.g., Seq ID 0102f02). As shown in Seq ID NO: 1, there is a 903 bp open reading frame (ORF) from 1426 bp to 2328 bp, encoding a new protein (as shown in Seq ID NO: 2). We named this clone pBS-0102f02, and the encoded protein human reverse transcription-associated factor 33. Example 2: Homology search of cDNA clones

[0083] The sequence of human reverse transcription-associated factor 33 and its encoded protein sequence of the present invention were analyzed using the Blast program (Basiclocal Alignment search tool) [Altschul, SF et al.]. [J.Mol. Biol.1990; 215: 403-10], homology searches were performed in databases such as Genbank and Swissport. The gene with the highest homology to human reverse transcription-associated factor 33 of this invention is a known L1 factor, whose encoded protein has access number 1109116 in Genbank. The protein homology results are shown in Figure 1, showing high homology with 90% identity and 94% similarity. Example 3: Cloning of the gene encoding human reverse transcription-associated factor 33 using RT-PCR.

[0084] Using total RNA from fetal brain cells as a template, cDNA was synthesized via reverse transcription using oligo-dT primers. After purification using a Qiagen kit, PCR amplification was performed using the following primers:

[0085] Primer 1: 5'-GAACTGAAGCTATCTATACATAAG —3, (SEQ ID NO: 3)

[0086] Primer2: 5'-AAAACATTCTTTATTTGGTCAATA - 3, (SEQ ID NO: 4)

[0087] Primerl is the forward sequence starting at the 5th lbp end of SEQ ID NO: 1;

[0088] Primer2 is the reversed 3-end sequence of SEQ ID NO: 1.

[0089] Amplification reaction conditions: A 50 μL reaction volume contained 50 mmol / L KCl, 10 mmol / L Tris-Cl (pH 8.5), 1.5 mmol / L MgCl2, 200 μmol / L dNTP, 10 pmol primer, and 1 U of Taq DNA polymerase (Clontech). The reaction was performed for 25 cycles on a PE9600 DNA thermal cycler (PerkinElmer) under the following conditions: 94°C for 30 sec; 55°C for 30 sec; 72°C for 2 min. β-actin was used as a positive control and template blank as a negative control during RT-PCR. The amplified products were purified using a QIAGEN kit and ligated into a PCR vector (Invitrogen) using a TA cloning kit. DNA sequence analysis showed that the DNA sequence of the PCR product was identical to 1-2707 bp as shown in SEQ ID NO: 1. Example 4: Northern blot analysis of human reverse transcription factor 33 gene expression:

[0090] Total RNA extraction using a one-step method [Anal. Biochem 1987, 162, 156-159] involves acidic guanidinium thiocyanate-phenol-chloroform extraction. Specifically, it uses 4M guanidinium isothiocyanate-25mM sodium citrate and 0.2M sodium acetate (…). PHomogenize the tissue using H4.0, add 1 volume of phenol and 1 / 5 volume of chloroform-isoamyl alcohol (49:1), mix and centrifuge. Aspirate the aqueous phase, add isopropanol (0.8 volume) and centrifuge the mixture to obtain RNA precipitate. Wash the obtained RNA precipitate with 70% ethanol, dry and dissolve in water. Use 20 μL... § RNA, in a solution containing 20 mM 3-(N-morpholino)propanesulfonic acid ( P Electrophoresis was performed on a 1.2% agarose gel containing 5mM sodium acetate, 1mM EDTA, and 2.2M formaldehyde. The gel was then transferred to a nitrocellulose membrane. (The text abruptly ends here, so the translation stops as well.) 32 P dATP was prepared using a random primer method. 32 P-labeled DNA probes. The DNA probes used were the PCR-amplified coding region sequence of human reverse transcription-related factor 33 (1426 bp to 2328 bp) shown in Figure 1. 32P-labeled probes (approximately 2 x 10⁻⁶) were then used. 6 (cpm / ml) Nitrocellulose membranes containing transferred RNA were hybridized overnight at 42°C in a solution containing 50% formamide - 25mM KH2PO4 (pH 7.4) - 5× SSC - 5× Denhardt's solution and 20 g / ml salmon sperm DNA. After hybridization, the membrane was filtered and incubated at 55°C in 1 SSC - 0.1% SDS. Wash for 30 min. Then, analyze and quantify using a Phosphor Imager. Example 5: In vitro expression, isolation, and purification of recombinant human reverse transcription-related factor 33.

[0091] Based on the coding region sequence shown in SEQ ID NO: 1 and Figure 1, a pair of specific amplification primers were designed, with the following sequences:

[0092] Primer 3: 5'-CATGCTAGCATGCTAGAAGAAAACCTAGGCAAT-3' (Seq ID No: 5) Primer 4: 5'-CCCGAGCTCTCAATTCCCACCTATGAGTGAGAA-3' (Seq ID No: 6) These two primers contain NH1 and Sacl restriction sites at their 5' ends, respectively, followed by the coding sequences for the 5' and 3' ends of the target gene. The NH1 and Sacl restriction sites correspond to the selective restriction enzyme sites on the expression vector plasmid pET-28b(+) (Novagen product, Cat. No. 69865.3). PCR was performed using the pBS-0102f02 plasmid containing the full-length target gene as a template. The PCR reaction conditions were as follows: a total volume of 50 μL containing 10 pg of pBS-0102f02 plasmid, 10 pmol each of primers Primer-3 and Primer-4, and 1 μL of Advantage polymerase Mix (Clontech). Cycling parameters: 94°C for 20 s, 60°C for 30 s, 68°C for 2 min, for a total of 25 cycles. The amplified product and plasmid pET-28(+) were double-digested with NH3 and SACIL, respectively, and the large fragments were recovered and ligated using T4 ligase. The ligation products were transformed into E. coli DH5α using the calcium chloride method. After overnight culture on LB agar plates containing kanamycin (final concentration 30 g / ml), positive clones were screened by colony PCR and sequenced. Positive clones with correct sequences (pET-0102f02) were selected, and the recombinant plasmid was transformed into *E. coli* BL21(DE3)plySs (Novagen) using the calcium chloride method. The host bacterium BL21 (pET-0102f02) was cultured in LB broth containing kanamycin (final concentration 30 g / ml) at 37°C to the logarithmic growth phase. IPTG was added to a final concentration of 1 μmol / L, and the culture was continued for 5 hours. The bacterial cells were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was then purified using a His. Bind Quick Cartridge (Novagen) affinity chromatography column that binds to six histidine residues (6His-Tag). The purified target protein, human reverse transcription factor 33, was obtained. SDS-PAGE electrophoresis showed a single band at 33 kDa (Figure 2).The band was transferred to a PVDF membrane and analyzed for its N-terminal amino acid sequence using the Edams hydrolysis method. The results showed that the N-terminal 15 amino acids were identical to the N-terminal 15 amino acid residues shown in SEQ ID NO: 2. Example 6: Generation of anti-human reverse transcription factor 33 antibody.

[0093] The following human reverse transcription-associated factor 33-specific polypeptide, NH2-Met-Leu-Glu-Glu-Asn-Leu-Gly-Asn-Thr-Ile-Gln-Asn-Ile-Gly-Met-C00H (SEQ ID NO: 7), was synthesized using a polypeptide synthesizer (PE company product). This polypeptide was then coupled with hemocyanin and bovine serum albumin to form complexes, as described in: Avrameas, et al. Immunochemistry, 1969; 6: 43. Rabbits were immunized with 4 mg of the above hemocyanin-polypeptide complex with complete Freund's adjuvant, and boosted with the hemocyanin-polypeptide complex with incomplete Freund's adjuvant after 15 days. Immunization was performed once. The antibody titer in the rabbit serum was determined by ELISA using a titration plate coated with a 15 g / ml bovine serum albumin-peptide complex. Total IgG was isolated from antibody-positive rabbit serum using Protein A-Sepharose. The peptide was bound to a cyanide-activated Sepharose 4B column, and anti-peptide antibodies were isolated from the total IgG by affinity chromatography. Immunoprecipitation demonstrated that the purified antibody specifically binds to human reverse transcription-associated factor 33. Example 7: Application of the polynucleotide fragment of the present invention as a hybridization probe.

[0094] Selecting suitable oligonucleotide fragments from the polynucleotides of the present invention for use as hybridization probes has many applications. For example, the probe can be used to hybridize with genomic or cDNA libraries of normal or pathological tissues from different sources to identify whether they contain the polynucleotide sequence of the present invention and to detect homologous polynucleotide sequences. Furthermore, the probe can be used to detect whether the expression of the polynucleotide sequence of the present invention or its homologous polynucleotide sequences is abnormal in normal or pathological tissue cells.

[0095] The purpose of this embodiment is to select suitable oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention as hybridization probes, and to identify whether some tissues contain the polynucleotide sequence of the present invention or its homologous polynucleotide sequence using a membrane hybridization method. Membrane hybridization methods include dot blot, Southern blotting, Northern blotting, and photocopying methods, all of which involve immobilizing the polynucleotide sample to be tested on a membrane and then hybridizing using essentially the same steps. These identical steps are: the membrane with the sample immobilized is first pre-hybridized with a hybridization buffer without probes to saturate the non-specific binding sites of the sample on the membrane with the carrier and the synthesized polymer. Then the pre-hybridization solution is replaced with a hybridization buffer containing labeled probes, and the membrane is incubated to allow the probes to hybridize with the target nucleic acid. After the hybridization step, the unhybridized probes are removed by a series of washing steps. This embodiment utilizes high-intensity washing conditions (such as low salt concentration and high temperature) to reduce the hybridization background and retain only highly specific signals. The probes used in this embodiment include two types: the first type of probe is an oligonucleotide fragment that is completely identical to or complementary to the polynucleotide SEQ ID NO: 1 of this invention; the second type of probe is an oligonucleotide fragment that is partially identical to or complementary to the polynucleotide SEQ ID NO: 1 of this invention. In this embodiment, dot blot is used to immobilize the sample on the filter membrane. Under high-intensity washing conditions, the first type of probe exhibits the strongest hybridization specificity with the sample and is thus retained.

[0096] I. Probe Selection

[0097] When selecting oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of this invention for use as hybridization probes, the following principles and several aspects should be followed:

[0098] 1. The preferred probe size range is 18-50 nucleotides;

[0099] 2. GC content should be 30%-70%; exceeding this range increases non-specific hybridization.

[0100] 3. There should be no complementary regions inside the probe;

[0101] 4. Probes meeting the above criteria can be used as initial selection probes, followed by further computer sequence analysis, including... The initial probe is compared for homology with its source sequence region (i.e., SEQ ID NO: 1) and other known genomic sequences and their complementary regions. If the homology with the non-target molecule region is greater than 85% or there are more than 15 consecutive identical bases, then the initial probe should generally not be used.

[0102] 5. Whether the initial probe is ultimately selected as a probe with practical application value should be further determined by experiments.

[0103] After completing the above analysis, the following two probes were selected and synthesized:

[0104] Probe 1, belonging to class 1 probes, is completely homologous to or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):

[0105] 5-TGCTAGAAGAAAACCTAGGCAATACATTCAGAACATAGGC-3' (SEQ ID NO: 8)

[0106] Probe 2, belonging to the second class of probes, is a substitutional mutant sequence (41Nt) corresponding to the gene fragment of SEQ ID NO: 1 or its complementary fragment:

[0107] 5'-TGCTAGAAGAAAACCTAGGCCATACCATTCAGAACATAGGC-3' (SEQ ID NO: 9) For other commonly used reagents not listed and their preparation methods related to the following specific experimental steps, please refer to: DNA PROBES GH Kel ler; MM Manak; Stockton Press, 1989 (USA) and more commonly used molecular cloning laboratory manuals such as Molecular Cloning: A Laboratory Manual (2nd edition, 1998) [US] Sambrook et al., Science Press.

[0108] Sample preparation:

[0109] 1. Extracting DNA from fresh or frozen tissues

[0110] Steps: 1) Place fresh or freshly thawed normal liver tissue into a petri dish containing phosphate-buffered saline (PBS) and place it on ice. Cut the tissue into small pieces with scissors or a scalpel. Keep the tissue moist during the process. 2) Centrifuge the tissue at 1000g for 10 minutes. 3) Resuspend the pellet in cold homogenization buffer (0. mol / L sucrose; mmol / L Tris-HCl, pH 7.5; 25 mmol / L NaCl; 25 mmol / L MgCl2) (approximately 10 ml / g). 4) Homogenize the tissue suspension at full speed using an electric homogenizer at 4°C until the tissue is completely broken down. 5) Centrifuge at 1000g for 10 minutes. 6) Resuspend the cell pellet (1-5 ml per 0.1g of the initial tissue sample) and centrifuge again at 1000g for 10 minutes. 7) Resuspend the precipitate with lysis buffer (1 ml for every 0.1 g of the initial tissue sample), and then proceed with the following phenol extraction method.

[0111] 2. Phenol extraction method for DNA

[0112] Steps: 1) Wash cells with 1-10 ml of cold PBS and centrifuge at 1000 g for 10 minutes. 2) Resuspend the precipitated cells in cold cell lysis buffer (1 x 10⁻⁶ cells / mL). 8 (Cells / ml) Use at least 100ul of lysis buffer. 3) Add SDS to a final concentration of 1%. If SDS is added directly to the cell pellet before resuspending the cells, the cells may form large clumps that are difficult to break, reducing the overall yield. This is especially important when extracting >10... 7 The effect is particularly severe in cells. 4) Add proteinase K to a final concentration of 200 μg / mL. 5) Incubate at 50°C for 1 hour or gently shake overnight at 37°C. 6) Extract with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and centrifuge for 10 minutes in a small centrifuge tube. The two phases should be clearly separated; otherwise, centrifuge again. 7) Transfer the aqueous phase to a new tube. 8) Extract with an equal volume of chloroform:isoamyl alcohol (24:1) and centrifuge for 10 minutes. 9) Transfer the aqueous phase containing DNA to a new tube. Then perform DNA purification and ethanol precipitation.

[0113] 3. DNA purification and ethanol precipitation

[0114] Steps: 1) Add 1 / 10 volume of 2mol / L sodium acetate and 2 volumes of cold 100% ethanol to the DNA solution and mix well. Incubate at -20°C for 1 hour or overnight. 2) Centrifuge for 10 minutes. 3) Carefully aspirate or pour off the ethanol. 4) Wash the precipitate with 500 μL of 70% cold ethanol and centrifuge for 5 minutes. 5) Carefully aspirate or pour off the ethanol. Wash the precipitate with 500 μL of cold ethanol and centrifuge for 5 minutes. 6) Carefully aspirate or pour off the ethanol, then invert the container on absorbent paper to allow any remaining ethanol to drain. Air dry for 10-15 minutes to allow surface ethanol to evaporate. Do not allow the precipitate to dry completely, otherwise it will be difficult to redissolve. 7) Resuspend the DNA precipitate in a small volume of TE or water. Vortex at low speed or use a dropper to aspirate, while gradually increasing the TE, mixing until the DNA is fully dissolved, 1-5 x 10⁻⁵ times per 10⁻⁶ ppm. 6 The amount extracted from the cells is approximately 1 ul.

[0115] Steps 8-13 below are only used when it is necessary to remove contaminants; otherwise, proceed directly to step 14.

[0116] 8) Add RNase A to the DNA solution to a final concentration of 100 μg / mL and incubate at 37°C for 30 minutes. 9) Add SDS and proteinase 1 to final concentrations of 0.5% and 100 μg / mL, respectively. Incubate at 37°C for 30 minutes. 10) Extract the reaction solution with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) and centrifuge for 10 minutes. 11) Carefully remove the aqueous phase and re-extract with an equal volume of chloroform:isoamyl alcohol (24:1) and centrifuge for 10 minutes. 12) Carefully remove the aqueous phase, add 1 / 10 volume of 2 mol / L sodium acetate and 2.5 volumes of cold ethanol, mix well, and incubate at -20°C for 1 hour. 13) Wash the precipitate with 70% ethanol and 100% ethanol, air-dry, and resuspend the nucleic acid, following steps 3-6. 14) Determine A 26 And A 2S To detect the purity and yield of DNA. 15) Store at -20°C after aliquoting.

[0117] Preparation of the sample membrane:

[0118] 1) Take 4 x 2 appropriately sized nitrocellulose membranes (NC membranes), and lightly mark the spotting location and sample number on them with a pencil. Two NC membranes are required for each probe so that the membranes can be washed under high-intensity conditions and low-intensity conditions respectively in the subsequent experimental steps.

[0119] 2) Take 15 μL of each sample and control sample, spot them onto the sample film, and let them air dry at room temperature.

[0120] 3) Place on filter paper soaked in 0.1 mol / L NaOH and 1.5 mol / L NaCl for 5 minutes (twice), then air dry.

[0121] 4) Place it between clean filter paper, wrap it with aluminum foil, and vacuum dry at 60-80°C for 2 hours.

[0122] Probe marking

[0123] 1) Add 3 μl Probe (0.10D / 10 μl), then add 2 μl Kinase buffer, and incubate with 8-10 uCi γ- 32 P_dATP+2U Kinase, to be added to a final volume of 20 μL. 2) Keep warm at 37°C for 2 hours.

[0124] 3) Add 1 / 5 volume of bromophenol blue indicator (BPB).

[0125] 4) Pass through Sephadex G-50 column.

[0126] 5) To have 32 The first peak is collected before the P-Probe wash (it can be monitored using a monitor).

[0127] 6) 5 drops / tube, collect 10-15 tubes.

[0128] 7) Monitor isotope levels using a liquid scintillation analyzer

[0129] 8) The collected liquid from the first peak is the desired product. 32 P-Probe (the second peak is free) Y - 32 P-dATP). Prehybridization

[0130] Place the sample membrane in a plastic bag, add 3-10 mg of prehybridization solution (10x Denhardt's; 6x SSC, 0.1 mg / ml CT DNA (calf thymus DNA)), seal the bag, and incubate at 68°C for 2 hours.

[0131] Hybridization

[0132] Cut off a corner of the plastic bag, add the prepared probe, seal the bag, and shake it in a 42°C water bath overnight.

[0133] Washing the mask:

[0134] High-intensity film washing:

[0135] 1) Take out the hybridized sample membrane.

[0136] 2) Wash at 40°C for 15 minutes (twice) in 2xSSC and 0.1%SDS.

[0137] 3) Wash at 40°C for 15 minutes (twice) in 0.1% SSC and 0.1% SDS.

[0138] 4) Wash in 0.1% SSC and 0.1% SDS at 55°C for 30 minutes (twice), then air dry at room temperature. Low-intensity washing:

[0139] 1) Remove the hybridized sample membrane...

[0140] 2) Wash in 2xSSC and 0.1%SDS at 37°C for 15 minutes (twice).

[0141] 3) Wash at 37°C for 15 minutes (twice) in 0.1% SSC and 0.1% SDS.

[0142] 4) Wash in 0.1% SSC and 0.1% SDS at 40°C for 15 minutes (twice), then air dry at room temperature.

[0143] X-ray autoradiography:

[0144] -70°C, X-ray autoradiography (the compression time depends on the intensity of the radioactivity of the hybridization spot).

[0145] Experimental results:

[0146] Hybridization experiments conducted under low-intensity washing conditions showed no significant difference in radioactivity between the two probe hybridization spots. However, hybridization experiments conducted under high-intensity washing conditions showed that the radioactivity intensity of the hybridization spot of probe 1 was significantly stronger than that of the other probe. Therefore, probe 1 can be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotides of this invention in different tissues. Example 8: Microarray

[0147] Gene chips, or DNA microarrays, are new technologies that many national laboratories and large pharmaceutical companies are currently researching and developing. They involve arranging a large number of target gene fragments in an orderly, high-density manner on a carrier such as glass or silicon, and then using fluorescence detection and computer software to compare and analyze the data, achieving rapid, efficient, and high-throughput analysis of biological information. The polynucleotides of this invention can be used as target DNA in gene chip technology for high-throughput research on the function of new genes; for finding and screening tissue-specific new genes, especially those related to diseases such as tumors; and for the diagnosis of diseases, such as hereditary diseases. Specific methods and steps have been reported in various literatures, such as DeRisi, LL., Lyer, V. & Brown, P.O.

[0148] (1997) Science 278, 680-686. and references Helle, RA, Schema, M., Chai, A., Shalom, D., (1997) PNAS 94: 2150-2155.

[0149] (a) Spotting

[0150] A total of 4000 polynucleotide sequences of various full-length cDNAs were used as target DNA, including the polynucleotides of this invention. These sequences were amplified separately by PCR. After purification, the amplified products were adjusted to a concentration of approximately 500 ng / µl and spotted onto a glass substrate using a Cartesian 7500 spotter (purchased from Cartesian, USA), with a spot spacing of 280 µm. The spotted slides were then hydrated, dried, and cross-linked in a UV cross-linker. After elution and drying, the DNA was immobilized on the glass slides to prepare a chip. Specific methods and steps have been reported in various literatures. The post-spotting processing steps in this embodiment are as follows:

[0151] 1. Hydrate for 4 hours in a humid environment;

[0152] 2. Wash with 0.2% SDS for 1 minute;

[0153] 3. Wash twice with ddH2O, 1 minute each time;

[0154] 4. NaBH4 seal for 5 minutes;

[0155] 5. In 95°C water for 2 minutes;

[0156] 6. Wash with 0.2% SDS for 1 minute;

[0157] 7. Rinse twice with ddH2O;

[0158] 8. Let it air dry, then store it in a dark place at 25°C for later use.

[0159] (II) Probe Marking

[0160] Total mRNA was extracted from normal liver and hepatocellular carcinoma using a one-step method, and the mRNA was purified using the Oligotex mRNA Midi Kit (QiaGen). Normal liver tissue mRNA was then labeled with the fluorescent reagent Cy3dUTP (5-Aminopropargyl-2-deoxyuridine 5-triphate coupled to Cy3 fluorescent dye, Amersham Phamacia Biotech) via reverse transcription. Cy5dUTP (5-Amino-propargyl-2'-deoxyuridine 5'-triphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech) was used to label liver cancer tissue mRNA, which was then purified to prepare the probe. For detailed steps and methods, please refer to:

[0161] Schena, M., Shalon, D., Hel ler, R. (1996) Proc. Natl. Acad. Sci. USA. Vol.93: 10614- 10619. Schena, M., Shalon, Dari., Davis, RW (1995) Science.270. (20): 467-480.

[0162] (III) Hybridization

[0163] The probes from the two tissues were hybridized with the chip in UniHyb™ Hybridization Solution (purchased from TeleChem) for 16 hours. After washing with washing solution (1 < SSC, 0.2% SDS) at room temperature, the samples were scanned using a ScanArray 3000 scanner (purchased from General Scanning, USA). The scanned images were processed using Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio for each point. Points with a ratio less than 0.5 and greater than 2 were considered to express differentially expressed genes. The experimental results showed that Cy3signal = 1267.64 (average of four experiments), Cy5signal = 7269.6 (average of four experiments), and Cy3 / Cy5 = 0.1744. The polynucleotides of the present invention showed significant differences in expression in the two tissues, indicating that the polynucleotides of the present invention are associated with liver cancer. Industrial Applicability

[0164] The polypeptides of the present invention, as well as their antagonists, agonists, and inhibitors, can be used directly for the treatment of diseases, such as malignant tumors, adrenal insufficiency, skin diseases, various inflammations, HIV infection, and immune diseases.

[0165] LRE2 on mammalian chromosome lq participates in reverse transcription. The proliferation of LRE2 involves three processes: transcription, reverse transcription, and reintegration into a new site on the genome. Insertion rearrangements at the LRE2 site can lead to abnormal gene expression, such as Duchenne muscular dystrophy. The polypeptide of this invention is homologous to LRE2 on mammalian chromosome lq and contains the characteristic sequence of the LRE2 family. It participates in reverse transcription in vivo, regulating the expression of certain genes. Abnormal expression of LRE2 can cause errors in the regulation of related genes and lead to related diseases.

[0166] Therefore, it is evident that abnormal expression of human reverse transcription-related factor 33 in this invention will lead to various diseases, especially muscular dystrophy, various arsenoblastomas, embryonic developmental disorders, growth and developmental disorders, inflammation, and immune diseases, including but not limited to:

[0167] Tumors in various tissues: stomach cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma. Thyroid tumors, uterine fibroids, neurocytomas, astrocytomas, ependymomas, gliomas, neurofibromas, colon cancer, melanomas, bladder cancer, uterine cancer, endometrial cancer, colon cancer, thymic tumors, nasopharyngeal carcinoma, laryngeal cancer, tracheal tumors, fibromas, fibrosarcomas, lipomas, liposarcomas

[0168] Embryonic developmental disorders: Congenital miscarriage, cleft palate, limb agenesis, limb differentiation disorders, atrial septal defect, neural tube defects, congenital hydrocephalus, congenital glaucoma or cataracts, congenital deafness.

[0169] Growth and developmental disorders: intellectual disability, brain development disorders, skin, fat and muscle dysplasia, bone and joint dysplasia, various metabolic disorders, cretinism, dwarfism, Cushing's syndrome, and sexual developmental delay.

[0170] Inflammation: Chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, multiple sclerosis of the cerebral and spinal cord, glomerulonephritis, myocarditis, cardiomyopathy, atherosclerosis, gastric ulcer, cervicitis, and various infectious inflammations.

[0171] Immunological diseases: Systemic lupus erythematosus, rheumatoid arthritis, bronchial asthma, urticaria, atopic dermatitis, post-infectious myocarditis, scleroderma, myasthenia gravis, Guillain-Barré syndrome, common variable immunodeficiency disease, primary B-lymphocyte immunodeficiency disease, acquired immunodeficiency syndrome.

[0172] Abnormal expression of human reverse transcription-related factor 33, as described in this invention, can also lead to certain hereditary and hematological diseases. The polypeptides of this invention, as well as their antagonists, agonists, and inhibitors, can be directly used for disease treatment, for example, to treat various diseases, especially muscular dystrophy, various tumors, embryonic developmental disorders, growth and developmental disorders, inflammation, immune diseases, and certain hereditary and hematological diseases.

[0173] This invention also provides a method for screening compounds to identify agents that enhance (agonist) or inhibit (antagonist) human reverse transcription-associated factor 33 (HRTF33). Agonists enhance HTF33's biological functions, such as stimulating cell proliferation, while antagonists inhibit and treat disorders associated with excessive cell proliferation, such as various cancers. For example, mammalian cells or membrane formulations expressing HTF33 can be co-cultured with labeled HTF33 in the presence of the drug. The ability of the drug to enhance or inhibit this interaction can then be determined.

[0174] Antagonists of human reverse transcription factor 33 include screened antibodies, compounds, receptor-deficient derivatives, and analogs. Antagonists of human reverse transcription factor 33 can bind to and eliminate its function, inhibit the production of the peptide, or bind to the active site of the peptide, preventing it from exerting its biological function.

[0175] When screening compounds for antagonism, human reverse transcription factor 33 (HRF3) can be added to a bioassay. The effect of a compound on the interaction between HRF33 and its receptor is measured to determine if the compound is an antagonist. Using the same method, receptor-deficient derivatives and analogs that act as antagonists can be screened. Peptide molecules that can bind to HRF3 can be... It is obtained by screening a random polypeptide library composed of various possible combinations of amino acids bound to a solid phase. During screening, human reverse transcription-related factor 33 molecules are generally labeled.

[0176] This invention provides a method for producing antibodies using polypeptides, fragments thereof, derivatives, analogs, or their cellular components as antigens. These antibodies may be polyclonal or monoclonal antibodies. This invention also provides antibodies against the human reverse transcription-associated factor 33 antigenic determinant. These antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, and fragments generated from Fab expression libraries.

[0177] Polyclonal antibodies can be produced by directly injecting human reverse transcription factor 33 into immunized animals (such as rabbits, mice, rats, etc.). Various adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant. Techniques for preparing monoclonal antibodies against human reverse transcription factor 33 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), three-cell hybridoma technology, human β-cell hybridoma technology, and EBV-hybridoma technology. Chimeric antibodies that combine human constant regions with non-human variable regions can be produced using existing techniques (Morrison et al., PNAS, 1985, 81: 6851). Existing techniques for producing single-chain antibodies (US Patent No. 4946778) can also be used to produce single-chain antibodies against human reverse transcription factor 33.

[0178] Antibodies against human reverse transcription factor 33 can be used in immunohistochemistry to detect human reverse transcription factor 33 in biopsy specimens.

[0179] Monoclonal antibodies that bind to human reverse transcription factor 33 can also be labeled with radioactive isotopes and injected into the body to track their location and distribution. These radiolabeled antibodies can serve as a non-invasive diagnostic method for locating tumor cells and determining whether metastasis has occurred.

[0180] Antibodies can also be used to design immunotoxins that target specific sites in the body, such as human reverse transcription factor.

[0181] 33. High-affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, and erythrine). A common method is to use a thiol cross-linking agent, such as SPDP, to attack the amino group of the antibody, binding the toxin to the antibody through disulfide bond exchange. Such hybrid antibodies can be used to kill human reverse transcription factor 33-positive cells.

[0182] The antibodies of this invention can be used to treat or prevent diseases associated with human reverse transcription factor 33. Administration of appropriate doses of the antibodies can stimulate or block the production or activity of human reverse transcription factor 33.

[0183] This invention also relates to diagnostic assays for the quantitative and localized detection of human reverse transcription factor 33 (HRTF33) levels. These assays are well known in the art and include FISH assays and radioimmunoassays. The HRTF33 levels detected in these assays can be used to interpret the importance of HRTF33 in various diseases and to diagnose diseases in which HRTF33 plays a role.

[0184] The peptides of the present invention can also be used for peptide profiling analysis. For example, the peptides can be specifically cleaved by physical, chemical or enzymatic methods and analyzed by one-dimensional, two-dimensional or three-dimensional gel electrophoresis, or more preferably by mass spectrometry. Polynucleotides encoding human reverse transcription factor 33 (HRTF33) can also be used for various therapeutic purposes. Gene therapy can be used to treat abnormal cell proliferation, development, or metabolism caused by the absence or abnormal / inactive expression of HRTF33. Recombinant gene therapy vectors (such as viral vectors) can be designed to express variants of HRTF33 to inhibit endogenous HRTF33 activity. For example, a variant of HRTF33 may be a shortened HRTF33 lacking the signal transduction domain; although it can bind to downstream substrates, it lacks signal transduction activity. Therefore, recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of HRTF33. Viral expression vectors such as retroviruses, adenoviruses, adenovirus-associated viruses, herpes simplex viruses, and parvoviruses can be used to transfer polynucleotides encoding HRTF33 into cells. Methods for constructing recombinant viral vectors carrying polynucleotides encoding human reverse transcription factor 33 can be found in existing literature (Sambrook, et al.). Furthermore, the recombinant polynucleotides encoding human reverse transcription factor 33 can be packaged into liposomes and transferred into cells.

[0185] Methods for introducing polynucleotides into tissues or cells include: directly injecting polynucleotides into tissues in vivo; or introducing polynucleotides into cells in vitro using vectors (such as viruses, bacteriophages, or plasmids) and then transplanting the cells into the body.

[0186] Oligonucleotides (including antisense RNA and DNA) and ribozymes that inhibit human reverse transcription-associated factor 33 raRNA are also within the scope of this invention. A ribozyme is an enzyme-like RNA molecule that specifically breaks down specific RNAs; its mechanism of action involves the ribozyme molecule specifically hybridizing with a complementary target RNA and then performing endonuclease. Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technology, such as the widely used technique of synthesizing oligonucleotides via solid-phase phosphoramide chemical synthesis. Antisense RNA molecules can be obtained by transcription of the DNA sequence encoding the RNA in vitro or in vivo. This DNA sequence is integrated downstream of the RNA polymerase promoter of the vector. To increase the stability of nucleic acid molecules, they can be modified in various ways, such as increasing the length of the flanking sequences, and using phosphothioester or peptide bonds instead of phosphodiester bonds for the ribonucleosides.

[0187] Polynucleotides encoding human reverse transcription factor 33 (HTF33) can be used for the diagnosis of diseases related to HTF33. These polynucleotides can be used to detect the expression of HTF33 or its abnormal expression in disease states. For example, the DNA sequence encoding HTF33 can be used to hybridize biopsy specimens to determine its expression status. Hybridization techniques include Southern blotting, Northern blotting, and in situ hybridization. These techniques are well-established and commercially available. Part or all of the polynucleotides of this invention can be used as probes immobilized on microarrays or DNA chips (also known as "gene chips") for differential gene expression analysis and gene diagnosis in tissues. In vitro amplification using HTF33-specific primers via RNA polymerase chain reaction (RT-PCR) is also possible. It can detect the transcription products of human reverse transcription-related factor 33.

[0188] Detecting mutations in the human reverse transcription factor 33 (RTF-33) gene can also be used to diagnose diseases related to RTF-33. RTF-33 mutations can take the form of point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human RTF-33 DNA sequence. Existing techniques such as Southern blotting, DNA sequencing, PCR, and in situ hybridization can be used to detect mutations. Furthermore, mutations may affect protein expression; therefore, Northern blotting and Western blotting can be used to indirectly determine the presence or absence of gene mutations.

[0189] The sequences of this invention are also valuable for chromosome identification. These sequences specifically target a particular location on a human chromosome and can hybridize with it. Currently, there is a need to identify the specific sites of genes on chromosomes. At present, only a few chromosome markers based on actual sequence data (repetitive polymorphisms) are available for labeling chromosome locations. According to this invention, a crucial first step in associating these sequences with disease-related genes is to locate these DNA sequences on chromosomes.

[0190] In short, PCR primers (preferably 15-35 bp) are prepared based on cDNA, allowing the sequence to be located on the chromosome. These primers are then used for PCR screening of somatic heterozygous cells containing each human chromosome. Only heterozygous cells containing the corresponding human genes from the primers will produce amplified fragments.

[0191] PCR localization of somatic hybrid cells is a rapid method for locating DNA to specific chromosomes. Using the oligonucleotide primers of this invention, sub-localization can be achieved using a set of fragments from a specific chromosome or a large number of genomic clones through a similar method. Other similar strategies that can be used for chromosome localization include in situ hybridization, chromosome pre-screening using labeled flow cytometry, and hybridization pre-selection, thereby constructing chromosome-specific cDNA libraries.

[0192] Fluorescence in situ hybridization (FISH) of cDNA clones with metaphase chromosomes allows for precise chromosome localization in a single step. For a review of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988).

[0193] Once a sequence is pinpointed to its precise chromosomal location, that physical location on the chromosome can be correlated with gene mapping data. This data can be found, for example, in V. McKusick, Mendelian Inheritance in Man (available via link to Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between the gene and diseases already located on the chromosomal region.

[0194] Next, it is necessary to determine the differences in cDNA or genomic sequences between affected and healthy individuals. If a mutation is observed in some or all affected individuals, but not in any healthy individuals, then that mutation may be the cause of the disease. Comparing affected and healthy individuals typically involves first looking for structural changes in chromosomes, such as deletions visible at the chromosomal level or detectable by cDNA sequence-based PCR. Or translocation. Based on the resolution of current physical mapping and gene localization technologies, cDNA that is precisely located to the chromosomal region associated with the disease can be one of 50 to 500 potential pathogenic genes (assuming a mapping resolution of 1 megabase and one gene per 20kb).

[0195] The peptides, polynucleotides and their analogues, agonists, antagonists and inhibitors of the present invention can be used in combination with suitable drug carriers. These carriers can be water, glucose, ethanol, salts, buffer solutions, glycerol and combinations thereof. The compositions contain a safe and effective amount of the peptide or antagonist, as well as a carrier and excipients that do not affect the efficacy of the drug. These compositions can be used as drugs for the treatment of diseases.

[0196] The present invention also provides a kit or reagent kit containing one or more containers, wherein the containers contain one or more pharmaceutical composition ingredients of the present invention. Along with these containers, there may be indicative instructions from the government regulatory authority that manufactures, uses, or sells the pharmaceutical or biological product, reflecting the authority's permission for its administration to humans. Furthermore, the polypeptides of the present invention can be used in combination with other therapeutic compounds.

[0197] The pharmaceutical composition can be administered in a convenient manner, such as via local, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, or intradermal routes. Human reverse transcription factor 33 is administered in an amount that is effective in treating and / or preventing a specific indication. The amount and dosage range of human reverse transcription factor 33 administered to a patient will depend on many factors, such as the route of administration, the patient's health condition, and the judgment of the diagnosing physician.

Claims

Claims I. An isolated polypeptide-human reverse transcription-related factor 33, characterized in that... It contains: a polypeptide of the amino acid sequence shown in SEQ ID NO: 2, or an active fragment of the polypeptide thereof, an analogue or a derivative thereof.

2. The polypeptide as described in claim 1, characterized in that... The amino acid sequence of the polypeptide, analogue or derivative has at least 95% identity with the amino acid sequence shown in SEQ ID NO:

2.

3. The polypeptide as described in claim 2, characterized in that... It contains a polypeptide having the amino acid sequence shown in SEQ ID NO:

2.

4. An isolated polynucleotide, characterized in that... The polynucleotide comprises one selected from the group consisting of: (a) a polypeptide or fragment thereof, analogue, or derivative thereof encoding the amino acid sequence shown in SEQ ID NO: 2; (b) Polynucleotides complementary to polynucleotides; or (c) A polynucleotide that is at least 91% identical to (a) or (b).

5. The polynucleotide as described in claim 4, characterized in that... The polynucleotide comprises a polynucleotide encoding the amino acid sequence shown in SEQ ID NO:

2.

6. The polynucleotide as described in claim 4, characterized in that... The polynucleotide sequence comprises the sequence of positions 1426-2328 in SEQ ID NO: 1 or the sequence of positions 1-2707 in SEQ ID NO:

1.

7. A recombinant vector containing exogenous polynucleotides, characterized in that... It is a recombinant vector constructed from the polynucleotide described in any one of claims 4-6 and a plasmid, virus, or vector expression vector.

8. A genetically engineered host cell containing exogenous polynucleotides, characterized in that... It is selected from one of the following host cells: (a) Host cells transformed or transduced using the recombinant vector of claim 7; or (b) Host cells transformed or transduced with the polynucleotide as described in any one of claims 4-6.

9. A method for preparing a polypeptide with human reverse transcription-associated factor 33 activity, characterized in that... The method includes: (a) The engineered host cells of claim 8 are cultured under conditions expressing human reverse transcription-related factor 33; (b) A polypeptide with human reverse transcription factor 33 activity was isolated from the culture.

10. An antibody capable of binding to a polypeptide, characterized in that... The antibody is an antibody that specifically binds to human reverse transcription-associated factor 33. II. A class of compounds that mimic or regulate the activity or expression of peptides, characterized in that... These are compounds that mimic, promote, antagonize, or inhibit the activity of human reverse transcription-associated factor 33.

12. The compound of claim 11, characterized in that... It is the antisense sequence of the polynucleotide sequence or a fragment thereof shown in SEQ ID NO:

1.

13. An application of the compound of claim 11, characterized in that... The compound is used for methods of regulating the in vivo and in vitro activity of human reverse transcription-related factor 33.

14. A method for detecting a disease or susceptibility to a disease associated with the polypeptide described in any one of claims 1-3, characterized in that... This includes detecting the expression level of the polypeptide, or detecting the activity of the polypeptide, or detecting nucleotide variations in the polynucleotide that cause abnormal expression or activity of the polypeptide.

15. The use of the polypeptide according to any one of claims 1-3, characterized in that... It is used to screen mimics, agonists, antagonists, or inhibitors of human reverse transcription-related factor 33; or for peptide fingerprint identification.

16. The application of the nucleic acid molecule according to any one of claims 4-6, characterized in that... It can be used as a primer for nucleic acid amplification reactions, or as a probe for hybridization reactions, or for manufacturing gene chips or microarrays.

17. The use of the polypeptide, polynucleotide, or compound according to any one of claims 1-6 and 11, characterized in that... The polypeptide, polynucleotide or its analogue, agonist, antagonist or inhibitor, in a safe and effective dose with a pharmaceutically acceptable carrier, constitutes a pharmaceutical composition for the diagnosis or treatment of diseases associated with abnormalities of human reverse transcription factor 33.

18. The use of the polypeptide, polynucleotide, or compound according to any one of claims 1-6 and 11, characterized in that... Drugs for treating conditions such as malignant tumors, hematological diseases, HIV infection, immune disorders, and various types of inflammation can be prepared using the aforementioned polypeptides, polynucleotides, or compounds.