WO2001075039A2 - A novel polypeptide, human rna helicase 10 and the polynucleotide encoding the polypeptide - Google Patents

Abstract

The present invention discloses a novel polypeptide, human RNA helicase 10, 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 malignant tumor, hemopathy, HIV infection, immunological disease, and various inflammation, 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 human RNA helicase 10.

Description

 A new peptide, human RNA helicase 10, and a polynucleotide encoding this polypeptide

 The present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide Λ RNA helicase 10, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. technical background

 In many activities of the cell, the regulation of RNA structure, such as splicing of precursor RNA, assembly of spliceosome, and protein translation, are all necessary. These processes are usually regulated by different RNA helicases. RNA helicases from different sources constitute a large family of proteins, and their presence can be detected in many biological systems where RM plays an important role. They are widely distributed in various tissues and organs from prokaryotes (including viruses) to lower and higher organisms, and are involved in cell and mitochondrial division, MA editing, rRNA processing, transcription initiation, nuclear mRNA transport, and mRNA Degradation and other processes. RNA helicases are considered to be important factors in cell development and differentiation, and some of them also play a role in the transcription and replication of viral single-stranded RNA [Arr i Ei sen, John C. Lucches i, Bioessays, 1998, 20: 634-641]. It provides effective means for the diagnosis, prevention and treatment of cancer, nervous system diseases and immune system diseases in vivo.

 RM helicases are divided into different subfamilies, subfamily I and subfamily Π according to their structural characteristics. All helicases contain two Walker-type NTP binding domains, the A-domain (or ATPase A domain) and the B-domain (or ATPase B domain). Members of subfamily I contain a conserved ATPase A domain: GXXXXGKT. Later, it was found that some ATPase domain A of some RNA helicases have mutations in some amino acid sites. The conserved sequence of the ATPase A domain is: AXXGXGKT, so these proteins are divided into one class, namely subfamily II . In subfamily II, some proteins contain a conserved DEAD box in the ATPase domain B. It was discovered that members of this subfamily can be divided into three different subclasses according to their DEAD boxes, namely DEAD boxes. Protein, DEAH box protein and DEXH box protein [Angel ika Luking, Ulf Stahl et al., 1998, Crit. Rev. Biochem. Mol. Biol., 33: 259-296].

In 1995, Zhang S. et al. Cloned the NDHI I protein from bovines, which is a member of the RM helicase family containing the DE box. It contains the DEIH motif in its domain II. This protein also has high homology with human helicase A and Drosophila Mle protein. All three are thought to play important roles in the metabolism of nuclear DM and MA [Zhang S., Maacke H. et al., 1995, J. Biol. Chem., 270: 16422-164 ² 7]. RM helicase A has both RM helicase activity and DM helicase activity in vivo. Therefore, the enzyme plays an important biological function in the transcriptional regulation of MA [Lee CG, Eki T et al., 1998, Genomi cs, 47: 365-371]. It can be known from the above that members of the RNA helicase protein family containing DEAD boxes play an important biological function in the transcriptional regulation of RNA, and the abnormal expression of such proteins will cause some metabolic disorders caused by abnormal transcriptional regulation A variety of immune system diseases and malignant diseases of some related tissues.

 Gene chip analysis revealed that in the thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, arsenic-stimulated L02 In cell lines and prostate tissues, the expression profile of the polypeptide of the present invention is very similar to the expression profile of human RNA helicase 95, so their functions may also be similar. The present invention was named human RM helicase 10.

 As mentioned above, the human RNA helicase 10 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, so there has been a need to identify more involved in these processes The human RM helicase 10 protein, especially the amino acid sequence of this protein was identified. The isolation of the newcomer MA helicase 10 protein encoding gene also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for the disease, so it is important to isolate its coding DNA. Object of the invention

 It is an object of the present invention to provide isolated new polypeptides-human RNA helicase 10 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 a human RNA helicase 10.

 Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human RM helicase 10.

 Another object of the present invention is to provide a method for producing human RNA helicase 10.

 Another object of the present invention is to provide an antibody against the polypeptide-human RNA helicase 10 of the present invention.

 Another object of the present invention is to provide analog compounds, antagonists, agonists, and inhibitors against the polypeptide-ΔRNA helicase 10 of the present invention.

Another object of the present invention is to provide diagnosis and treatment of diseases related to abnormalities in human MA helicase 10 Methods. Summary of invention

 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. Preferably, 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:

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

 (b) a polynucleotide complementary to the polynucleotide (a);

 (c) A polynucleotide having at least 70% identity to a polynucleotide sequence of (a) or (b).

 More preferably, the sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 738 to 1013 in SEQ ID NO: 1; and (b) a sequence having 1-2421 in SEQ ID NO: 1 Sequence of bits.

 The present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.

 The invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.

 The invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of human RNA helicase 10 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 RNA helicase 10 protein in vitro, which comprises detecting a mutation in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, or detecting a biological The amount or biological activity of a polypeptide of the invention in a sample.

 The invention also relates to 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 RNA helicase 10.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein. BRIEF DESCRIPTION OF THE DRAWINGS

 The following drawings are used to illustrate specific embodiments of the present invention, but not to limit the scope of the present invention as defined by the claims.

 Fig. 1 is a comparison diagram of gene chip expression profiles of human RNA helicase 10 and human RNA helicase 95 of the present invention. The upper graph is a graph of the expression profile of human RNA helicase 10, and the lower graph is the graph of the expression profile of human RM helicase 95.

 Figure 2 is a polyacrylamide gel electrophoresis image (SDS-PAGE) of the isolated human RNA helicase 1G. 1 OkDa is the molecular weight of the protein. The arrow indicates the isolated protein band. Summary of the Invention

 The following terms used in this specification and the claims shall have the following meanings unless specifically stated: "Nucleic acid sequence" refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and may also refer to the genome or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand. Similarly, the term "amino acid sequence" refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof. When the "amino acid sequence" in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide" or "protein" does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .

 A "variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. Variants can have "conservative" changes, in which the amino acid substituted 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" or "addition" means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature. "Replacement" refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.

 "Biological activity" refers to a protein that has the structure, regulation, or biochemical function of a natural molecule. Similarly, the term "immunologically active" refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.

An "agonist" refers to a molecule that, when combined with human RM helicase 10, causes a change in the protein to regulate the activity of the protein. Agonists can include proteins, nucleic acids, carbohydrates or any Any other molecule that can bind human RNA helicase 10.

 An "antagonist" or "inhibitor" refers to a molecule that, when combined with human MA helicase 10, can block or modulate the biological or immunological activity of human RNA helicase 10. Antagonists and inhibitors can include proteins, nucleic acids, carbohydrates or any other molecule that can bind to human RM helicase 10.

 "Regulation" refers to a change in the function of human RNA helicase 10, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological, functional, or immune properties of human RNA helicase 10.

 "Substantially pure" means substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. Those skilled in the art can purify human MA helicase 10 using standard protein purification techniques. Essentially pure human RM helicase 10 produces a single main band on a non-reducing polyacrylamide gel. The purity of the human MA helicase 10 polypeptide can be analyzed by amino acid sequence.

 "Complementary" or "complementary" refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature. For example, the sequence "C-T-G-A" can be combined with the 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. This inhibition of hybridization can be detected by performing hybridization (Southern imprinting or Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.

 "Percent identity" refers to the percentage of sequences that are the same or similar in a comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as through the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Clus ter method (Hi ggins, DG and PM Sharp (1988) Gene 73: 237-244). The C lus ter method checks the distance between all pairs The groups of sequences are arranged into clusters. The clusters are then allocated in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by

 Number of residues matching between sequence ^ and sequence ^

Number of residues in sequence-number of interval residues in sequence ^-number of interval residues in sequence ^X

Nucleic acid sequences can also be determined by the Clus ter method or by methods known in the art such as: Totun Hein Percentage identity (Hein J., (1990) Methods in enzymology 183: 625-645) ₀ "Similarity" refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences . Amino acids used for conservative substitution, 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 strand that is complementary to a "sense strand."

 "Derivative" refers to HFP or a chemical modification of its nucleic acid. 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,? (^ ') ₂ and?, Which can specifically bind to the epitope of human RNA helicase 10.

 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.

 The term "isolated" refers to the removal of a substance from its original environment (for example, its natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, 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 part of its natural environment, they are still isolated.

 As used herein, "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). For example, 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 .

 As used herein, "isolated human RNA helicase 10" means that human RNA helicase 10 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify human RNA helicase 10 using standard protein purification techniques. Substantially pure polypeptides produce a single main band on a non-reducing polyacrylamide gel. The purity of the human RM helicase 10 peptide can be analyzed by amino acid sequence.

The present invention provides a novel polypeptide-human MA helicase 10, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2. The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, A recombinant polypeptide is preferred. 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 RNA helicase 10. As used in the present invention, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially maintains the same biological function or activity of the human RNA helicase 10 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 (Π) a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or (Π Ι) Such a polypeptide in which the mature polypeptide is fused to another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV) · A polypeptide in which an additional amino acid sequence is fused to the mature polypeptide Sequence (such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protein sequence) As explained herein, such fragments, derivatives, and analogs are considered to be within the knowledge of those skilled in the art.

 The present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2. The polynucleotide sequence of the present invention includes 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 full-length polynucleotide sequence of 2421 bases, and its open reading frame 738-1013 encodes 91 amino acids. According to the comparison of gene chip expression profiles, it was found that this polypeptide has a similar expression profile with human RNA helicase 95, and it can be deduced that the human RNA helicase 10 has similar functions to human RM helicase 95.

 The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be coding or non-coding. The coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant. As used herein, a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.

 The polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.

The term "polynucleotide encoding a polypeptide" is meant to include polynucleotides that encode such polypeptides and polynucleotides that include additional coding and / or noncoding 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 may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .

 The invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity, between the two sequences). The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "strict conditions" means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficol 1, 42 ° C, etc .; or (3) only between the two sequences Hybridization occurs only when the identity is at least 95%, and more preferably 97%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.

 The invention also relates to nucleic acid fragments that hybridize to the sequences described above. As used in the present invention, a "nucleic acid fragment" contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nuclei. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding human RNA helicase 10.

 The 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 RM helicase 10 of the present invention can be obtained by various methods. For example, polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.

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

Of the methods mentioned above, 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 niRNA 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 extracting mRNA, and kits are also commercially available (Qiagene). And the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manua, Cold Spruing Harbor Laboratory. New York, 1989). Commercially available cDNA libraries are available, such as different cDNAs from Clontech W 01 Library. When polymerase reaction technology is used in combination, even very small expression products can be cloned.

 The genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DM-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) measuring the level of human RNA helicase 10 transcripts; (4) ) Detection of protein products expressed by genes through immunological techniques or determination of biological activity. The above methods can be used singly or in combination.

 In the 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, preferably at least 100 nucleotides. In addition, the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides. The probe used here is usually 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. DM probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).

 In the (4) method, immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein products expressed by the human RNA helicase 10 gene.

 A method (Sa iki, et al. Sc; 1985; 230: 1350-1354) using PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention. In particular, when it is difficult to obtain a full-length cDNA from a library, the RACE method (RACE-Rapid Amplification of cDNA Ends) can be preferably used. The primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein Select and synthesize using conventional methods. The amplified DNA / RM fragments can be isolated and purified by conventional methods such as by gel electrophoresis.

 The 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, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.

 The present invention also relates to a vector comprising a polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a human RNA helicase 10 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology. .

In the present invention, a polynucleotide sequence encoding human RNA helicase 10 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention. The term "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, 56: 125) expressed in bacteria; pMSXND expression vectors expressed in mammalian cells (Lee and Na thans, J Bi o Chem. 263: 3521, 1988) And baculovirus-derived vectors expressed in insect cells. In short, as long as it can be replicated and stabilized in a host, any plasmid and vector can be used to construct a recombinant expression vector. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.

 Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human RNA helicase 10 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, integrative synthesis technology, and in vivo recombination technology (Sambroook, et al. Mole ecu lar Cloning, a Laboratory Manua, Cold Spring Harbor Laboratory. New York, 1989). The DNA sequence can be operably linked to an appropriate promoter in the expression vector to guide oiRM synthesis. Representative examples of these promoters are: the lac or trp promoter of E. coli; the PL promoter of lambda phage; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, Retroviral LTRs and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation, a transcription terminator, and the like. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors expressed by DM, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers and adenovirus enhancers on the late side of the origin of replication.

 In addition, 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. Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

 Those of ordinary skill in the art will know how to select appropriate vector / transcription control elements (such as promoters, enhancers, etc.) and selectable marker genes.

 In the present invention, a polynucleotide encoding human MA helicase 10 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector. The term "host cell" refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E. coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as fly S2 or Sf9; animal cells such as CH0, COS or Bowes melanoma cells.

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. When the host is a prokaryote, such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl ₂ method. The steps used are well known in the art. Alternatively, MgCl ₂ is used. If required, transformation can also be performed by electroporation Method. When the host is a eukaryote, the following DM transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.

 Using conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant human RNA helicase 10 (Science, 1984; 224: 1431). Generally there are the following steps:

(1) using a polynucleotide (or variant) encoding human human RNA helicase 10 of the present invention, or transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide;

 (2) culturing host cells in a suitable medium;

 (3) Isolate and purify protein from culture medium or cells.

 In step (2), depending on the host cell used, 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.

 In step (3), the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

 The polypeptides of the present invention, as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.

 RNA and DNA helicases are expressed in cancer tissues, brain and nervous tissues, and tissues associated with inflammation and immune responses. Therefore, RNA helicases play an important role in cancer, nervous system diseases, and immune system diseases. In particular, the expression of RNA helicase is significantly increased in cancer and immune system diseases; in nervous system-related diseases, its expression level or activity is reduced.

 Specifically, according to the novel human MA helicase of the present invention, the enzyme and its fragments or derivatives can be used to diagnose and treat diseases related to the nervous system, including but not limited to the following, sedation panic disorder, premature senility Dementia, memory anterograde amnesia, amyotrophic lateral sclerosis, bipolar neurocytic disorder, tension, brain tumors, dementia, depression, delayed dyskinesia, dystonia, epilepsy, hereditary chronic chorea, multiple Lateral sclerosis, neurofibromas, Parkinson's disease, paranoid neuropathy, schizophrenia, Tourette's disease, etc.

The novel helicase and its fragments or derivatives of the present invention can also be used to diagnose and treat some cancers These cancers include but are not limited to the following: adenoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, etc .; in particular, cancers including the following tissues, thyroid, bladder, bone, bone marrow, brain, breast , Cervical, tendon sheath cyst, heart, kidney, lung, liver, muscle, ovary, pancreas, parathyroid gland, prostate, uterus, salivary gland, skin, spleen, testis, penis, thymus, gallbladder, gastrointestinal tract, etc.

 The novel human helicases and fragments or derivatives thereof of the present invention can also be used to diagnose and treat diseases related to the immune system, including but not limited to the following, rheumatoid arthritis, chronic active hepatitis, primary Sjogren's syndrome, ankylosing spondylitis, hemochromatosis, immune complex glomerulonephritis, mycocarditis after gonococcal infection, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, polymyositis, mouth Xerophthalmia syndrome, nodular polyarteritis, Wegener's granulomatosis, myasthenia gravis, Guillain-Barre syndrome, autoimmune hemolytic anemia, immune thrombocytopenic purpura, insulin autoimmune syndrome, autoimmune Immune thyroid disease, autoimmune heart disease, Down syndrome, short limb dwarfism, hereditary transcobalamin II deficiency with hypogammaglobulinemia, biotin-dependent carboxylase deficiency, Dun Can syndrome , Thymoma, chronic disease skin mucosal candidiasis, aplastic anemia, Di George syndrome, Wiscot t -Aldr ich syndrome, immunodeficiency disease with ataxia telangiectasia, etc.

 The novel human helicases and fragments or derivatives thereof of the present invention can also be used to diagnose and treat diseases related to the immune system, including but not limited to the following, pulmonary eosinophilia, sarcoidosis, rheumatism Arthritis, rheumatoid arthritis, osteoarthritis, cholecystitis, glomerulonephritis, immune complex glomerulonephritis, acute anterior uveitis, osteoporosis, dermatomyositis, urticaria, Specific dermatitis, hemochromatosis, polymyositis, Addison's disease, Graves' disease, chronic active hepatitis, emergency bowel syndrome, atrophic gastritis, systemic lupus erythematosus, myasthenia gravis, cerebral spinal cord Multiple sclerosis, Guillain-Barre syndrome, intracranial granuloma, Wegener's granulomatosis, autoimmune thyroiditis, autoimmune interstitial nephritis, ulcerative colitis, anemia, pancreatitis, segmental ileitis, Myocarditis, atherosclerosis, multiple scleroderma, etc.

 The invention also provides methods of screening compounds to identify agents that increase (agonist) or suppress (antagonist) human RNA helicase 10. Agonists enhance human RNA helicase 10 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers. For example, mammalian cells or membrane preparations expressing human RNA helicase 10 can be cultured with labeled human RM helicase 10 in the presence of drugs. The ability of the drug to increase or block this interaction is then determined.

Antagonists of human RNA helicase 10 include screened antibodies, compounds, receptor deletions, and the like. Antagonists of human RM helicase 10 can bind to human RNA helicase 10 and eliminate its function, Either the production of the polypeptide is inhibited or the active site of the polypeptide is combined so that the polypeptide cannot perform a biological function.

 When screening compounds as antagonists, human RM helicase 10 can be added to bioanalytical assays to determine whether a compound is an antagonist by measuring the effect of the compound on the interaction between human RM helicase 10 and its receptor. . Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds. Polypeptide molecules capable of binding to human RNA helicase 10 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, generally 10 molecules of human RM helicase should be labeled.

 The present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies. The invention also provides antibodies against human RM helicase 10 epitopes. These antibodies include (but are not limited to): Doklon 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 RNA helicase 10 directly into immunized animals (such as rabbits, mice, rats, etc.). A variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant. Wait. Techniques for preparing monoclonal antibodies to human RNA helicase 10 include, but are not limited to, hybridoma technology (Kohler and Mistein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma technology, EBV-hybridoma technology, etc. Chimeric antibodies that bind human constant regions to non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851). ₀ Existing techniques for producing single-chain antibodies (US Pat No. 4946778) can also be used to produce single chain antibodies against human RM helicase 10.

 Antibodies to human RNA helicase 10 can be used in immunohistochemistry to detect human RM helicase 10 in biopsy specimens.

 Monoclonal antibodies that bind to human RM helicase 10 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.

 Antibodies can also be used to design immunotoxins that target a particular part of the body. For example, human RM helicase 10 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 RM helicase 10 positive cells .

The antibodies in the present invention can be used to treat or prevent human MA helicase 10-related diseases. Give An appropriate dose of the antibody can stimulate or block the production or activity of human RNA helicase 10.

 The invention also relates to a diagnostic test method for quantitative and localized detection of human RNA helicase 10 levels. These tests are well known in the art and include FISH assays and radioimmunoassays. The level of human RNA helicase 10 detected in the test can be used to explain the importance of human RNA helicase 10 in various diseases and to diagnose diseases in which human MA helicase 10 functions.

 The polypeptide of the present invention can also be used for peptide mapping analysis. For example, 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 RNA helicase 10 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 RNA helicase 10. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutant human MA helicase 10 to inhibit endogenous human RM helicase 10 activity. For example, a mutant human MA helicase 10 may be a shortened human RNA helicase 10 lacking a signaling domain, and although it can bind to a downstream substrate, it lacks signaling activity. Therefore, recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of human RNA helicase 10. 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 RM helicase 10 into cells. Methods for constructing recombinant viral vectors carrying a polynucleotide encoding human RNA helicase 10 can be found in the existing literature (Sambrook, et al.). Alternatively, a recombinant polynucleotide encoding human RNA helicase 10 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.

 Oligonucleotides (including antisense RNA and DNA) and ribozymes that inhibit human RM helicase 10 mRNA are also within the scope of the present invention. A ribozyme is an enzyme-like RNA molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RM to perform endonucleation. Antisense RNA, DM, and ribozymes can be obtained by any of the existing RNA or DM synthesis techniques, such as solid-phase phosphate amide chemical synthesis to synthesize oligonucleotides. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of the DM sequence encoding the A. This DNA sequence has been integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphorothioate or peptide bonds instead of phosphodiester bonds.

Polynucleotides encoding human RNA helicase 10 are useful in diseases related to human RM helicase 10 Diagnosis. The polynucleotide encoding human RNA helicase 10 can be used to detect the expression of human RNA helicase 10 or the abnormal expression of human RNA helicase 10 in a disease state. For example, the DM sequence encoding human RNA helicase 10 can be used to hybridize biopsy specimens to determine the expression status of human RM helicase 10. Hybridization techniques include Southern blotting, Northern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available. A part or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray (Microarray) or a DM chip (also known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues. Human RNA helicase 10 specific primers can also be used to detect the transcription products of human RNA helicase 10 by MA-polymerase chain reaction (RT-PCR) in vitro amplification.

 Detection of mutations in the human RNA helicase 10 gene can also be used to diagnose human RNA helicase 10-related diseases. Human RNA helicase 10 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type human RNA helicase 10 DNA sequences. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.

 The sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.

 In short, PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.

 PCR localization of somatic hybrid cells is a quick way to localize DM to specific chromosomes. Using the oligonucleotide primers of the present invention, in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization. Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct chromosome-specific cDM libraries.

 Fluorescent in situ hybridization (FISH) of cDM clones with metaphase chromosomes allows precise chromosomal localization in one step. For a review of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Pres s, New York (1988).

Once the sequence is located at the exact chromosomal location, the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in V. Mckusick, Mendel ian Inher i tance in Man (available online with Johns Hopkins University Welch Medica l Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.

 Next, the difference in cDM 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. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).

 The polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier. These carriers 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. Along with 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. In addition, the polypeptides of the invention can be used in combination with other therapeutic compounds.

 The pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration. Human RNA helicase 10 is administered in an amount effective to treat and / or prevent a specific indication. The amount and range of human RNA helicase 10 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. Examples

 The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods in the following examples are not marked with specific conditions, usually according to conventional conditions, such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Harbor Harbor Laboratory Pres s, 1989), or according to the conditions described in Conditions recommended by the manufacturer. ,

 Example 1 Cloning of human RNA helicase 10

Total RM of human fetal brain was extracted by one step method with guanidine isothiocyanate / phenol / chloroform. Quik mRNA Isolat ion Ki t (Qiegene). Isolate poly (A) mMA from total RNA. 2ug poly (A) mRNA is reverse transcribed to form cDNA. Smart cDM cloning kit (purchased from Clontech ^ cDNA fragment was inserted into the multiple cloning site of pBSK (+) vector (Clontech)) to transform DH5 α to form a cDNA library. Dye terminate cycle react ion sequencing ki t (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer) determine the 5 'and 3' ends of all clones. The determined cDNA sequences were compared with the existing public DNA sequence database (Genebank). By comparison, it was found that the CDM sequence of one of the clones 0471a03 was a new DM. A series of primers were synthesized to determine the inserted cDNA fragment of the clone in both directions. The results showed that the full length CDM of the 0471a03 clone was 24 ² lbp ( As shown in Seq ID N0: l), there is a 275bp open reading frame (0RF) from 738bp to 1013bp, which encodes a new protein (as shown in Seq ID NO: 2). We named this clone pBS- 0471a03, the encoded protein is named human RNA helicase 10. Example 2 The gene encoding human RNA helicase 10 was cloned by RT-PCR method

 CDNA was synthesized using fetal brain total RM 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:

 Primerl: 5, one ATCTTCACCCAGAGGAATACTATG-3 '(SEQ ID NO: 3)

 Primer 2: 5'- TTCTGTATTTTGTAGAGGTGGGGT-3 '(SEQ ID NO: 4)

 Pr imerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;

Primer ² is the 3 'end reverse sequence in SEQ ID NO: 1.

Amplification conditions: 50 _μl / LC l, 10 mmol / L Tris-HCl, pH 8.5, 1.5 mmol / L MgCl ₂ , 20 (^ mol / L dNTP, lOpmol primer, 1U Taq DNA polymerase (product of Clontech). Reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elraer) for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min. Set β-act in as positive control and template blank as negative control at the same time during RT-PCR. The amplified product was purified with QIAGEN's kit and connected to pCR vector with TA cloning kit (Invitrogen). DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as l-2421bp shown in SEQ ID NO: 1. Example 3 Northern RNA analysis of human RNA helicase 10 gene expression

Total RNA extraction in one step [Anal. Biochem 1987, 162, 156-159]. This method involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1) Centrifuge after mixing. The aqueous layer was aspirated, isopropanol (0.8 vol) was added and the mixture was centrifuged to obtain RM precipitate. Will The obtained RM precipitate was washed with 70% ethanol, dried and dissolved in water. Performed with 2 g RNA on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (H7. 0)-5 mM sodium acetate-ImM EDTA-2. 2M formaldehyde electrophoresis then transferred to nitrocellulose by c -. ³² P dATP labeled probe ³² P- DM prepared by the random primer DM SYSTEM probe used is shown in FIG PCR amplified human RNA helicase. Enzyme 10 coding region sequence (738bp to 1013bp). 32P-labeled probe (about 2 x 10 ⁶ cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred in a solution at 42 ° C overnight. The solution Contains 50% formamide-25mM KH ₂ P0 ₄ (pH7. 4) -5 χ SSC-5 χ Denhardt's solution and 20 (g / ml salmon sperm DNA. After hybridization, the filter is placed at 1 x SSC- 0.1% Wash the SDS at 55 ° C for 30rain. Then, use Phosphor Imager for analysis and quantification. Example 4 In vitro expression, isolation, and purification of recombinant human RNA helicase 10

 Based on SEQ ID NO: 1 and the coding region sequence shown in Figure 1, a pair of specific amplification primers was designed, the sequence is as follows:.

Pr imer3: 5 '-CCCCATATGATGCATGAAGTATGTTCTGAGCAC-3' (Seq ID No: 5) Priraer4: 5'-CATGGATCCTTATATGACTAGAAACAGGAGGGA-3 '(Seq ID No: 6) The 5' ends of these two primers contain Ndel and BainHI restriction sites, respectively , Followed by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively, and the Ndel and BamHI digestion sites correspond to the expression vector plasmid pBT-28b (+) (Novagen, Ca. No. 69865. 3) Selective endonuclease site. PCR was performed using the PBS-0471a 03 plasmid containing the full-length target gene as a template. PCR reaction conditions were: 1 in a total volume of ₅₀ μ plasmid pBS-0471a03 containing 10pg, primers Pr imer-3 and Pr imer-4 were l Opmol, Advantage polymerase Mix (Clontech Products) 1μ1. Cycle parameters: 94 ° C 20s, 60. C 30s, 68 ° C 2 min, a total of 25 cycles. Ndel 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. Ligation products were transformed by the calcium chloride method Escherichia bacteria DH5 a, the (final concentration of 30μ ₈ / ιη1) LB plates incubated overnight positive clones by colony PCR method containing kanamycin, and sequenced. A positive clone (pBT-0471a03) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method. In containing kanamycin (final concentration ₃₀ μ§ / ηι1) in LB liquid medium, host strain BL21 _(P ET-0471a03) incubated at 37 ° C to logarithmic phase, IPTG was added to a final concentration of lmmol / L , Continue to cultivate for 5 hours. The cells were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. The affinity chromatography column His s. Bind Quick Cartr idge (product of Novagen) was used to bind 6 histidines (6His-Tag). By chromatography, the purified human protein helicase 10 was obtained. After SDS-PAGE electrophoresis, a single band was obtained at 10 kDa (Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by Edams hydrolysis method. As a result, the 15 amino acids at the N-terminus were identical to the 15 amino acid residues at the N-terminus shown in SEQ ID NO: 2. Example 5 Production of anti-human MA helicase 10 antibody

 The following peptides specific for human RNA helicase 10 were synthesized using a peptide synthesizer (product of PE):

NH2-Met-His-Glu-Val-Cys-Ser-Glu-His-Leu-Ser-Trp-Phe-Arg-Arg-Cys-C00H (SEQ ID NO: 7). The polypeptide was coupled to hemocyanin and bovine serum albumin to form a complex, respectively. For methods, see: Avrameas, et al. Immunochemi s try, 1969; 6: 43. Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once. A titer plate coated with 15 _g / ml bovine serum albumin peptide complex was used as an ELISA to determine the antibody titer in rabbit serum. Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose. The peptide was bound to a cyanogen bromide-activated 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 MA helicase 10. Example 6 Application of the polynucleotide fragment of the present invention as a hybridization probe

 The suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in various aspects. For example, the probes can be used to hybridize to the genome or CDM library 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. Further, 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 using a filter hybridization method. Acid sequence or a homologous polynucleotide sequence thereof. 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. These same steps are as follows: 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 the labeled probe and incubated to hybridize the probe to the target nucleic acid. After the hybridization step, 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. In this embodiment, 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. First, the selection of the probe

 The selection of 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:

 1. The preferred range of probe size is 18-50 nucleotides;

 2, GC content is 30% -70%, non-specific hybridization increases when it exceeds;

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

 4. 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 For homology comparison of the regions, 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 generally;

 5. Whether the preliminary selection probe is finally selected as a probe with practical application value should be further determined by experiments.

 After completing the above analysis, select and synthesize the following two probes:

 Probe 1 (probel), which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):

 5'-TGCATGAAGTATGTTCTGAGCACCTTTCCTGGTTCAGGCGT-3, (SEQ ID NO: 8) Probe 2 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutant sequence of the gene fragment of SEQ ID NO: 1 or its complementary fragment (41Nt):

 5'-TGCATGAAGTATGTTCTGAGCACCTTTCCTGGTTCAGGCGT-3 '(SEQ ID NO: 9) For other commonly used reagents and their preparation methods not related to the following specific experimental steps, please refer to the literature: DNA PROBES G. Η · Keller; MM Manak; Stockton P. ress, 1989 (USA) and more commonly used molecular cloning laboratory manuals such as "Molecular Cloning Experiment Guide" (Second Edition 1998) [US] Sambrook et al., Science Press.

 Sample Preparation:

 1.Extract DNA from fresh or frozen tissue

Procedure: 1) Place fresh or freshly thawed normal liver tissue in a plate immersed in ice and filled with phosphate buffered saline (PBS). Cut the tissue into small pieces with scissors or a scalpel. Keep tissue moist during operation. 2) Centrifuge the tissue at 1,000 g for 10 minutes. 3) Suspend the precipitate (about 10 ml / g) with cold homogenization buffer (0.25 mol / L sucrose; 25 ol / L Tris-HCl, pH 7.5; 25 fmol / L NaCl; 25 mmol / L MgCl ₂ ). 4) at 4. C Use an electric homogenizer to homogenize the tissue suspension at full speed until the tissue is completely broken. 5) Centrifuge at 1000g for 10 minutes. 6) Resuspend the cell pellet (l-5 ml per 0.1 g of the original tissue sample), and centrifuge at 1,000 g for 10 minutes. 7) Resuspend the pellet in lysis buffer (add 1 ml per 0.1 g of the initial tissue sample), then connect the following Phenol extraction method.

 2, DNA phenol extraction method

Procedure: 1) Wash cells with 1-lQml cold PBS and centrifuge at 1GOOg for 10 minutes. 2) Resuspend the pelleted cells with cold cell lysate (1 x ^{10 8} cells / ml) and apply a minimum of 100 ul of lysis buffer. 3) Add SDS to a final concentration of /. If SDS is added directly to the cell pellet before resuspending the cells, the cells may form large clumps that are difficult to break, and reduce the overall yield. This is particularly serious when extracting> 10 ⁷ cells. 4) Add proteinase K to a final concentration of 200ug / ml. 5) Incubate at 50 ° C for 1 hour or shake gently at 37 ° C overnight. 6) Extract with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and centrifuge in a small centrifuge tube for 10 minutes. The two should be clearly separated, otherwise centrifuge again. 7) Transfer the water 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 DNA-containing aqueous phase to a new tube. Purification of DM and ethanol precipitation were then performed.

 3, DNA purification and ethanol precipitation

Steps: 1) Add 1/10 volume of 2mol / L sodium acetate and 2 volumes of cold 100% ethanol to the DNA solution and mix. At -20. C Let stand for 1 hour or overnight. 2) Centrifuge for 10 minutes. 3) Carefully aspirate or pour out the ethanol. 4) Wash the pellet with 500ul of 70% cold ethanol and centrifuge for 5 minutes. 5) Carefully aspirate or pour out the ethanol. Wash the pellet with 500ul of cold ethanol and centrifuge for 5 minutes. 6) Carefully aspirate or pour out the ethanol, then invert on the absorbent paper to drain off the residual ethanol. Air dry for 10-15 minutes to allow the surface ethanol to evaporate. Be careful not to allow the pellet to dry completely, otherwise it will be more difficult to re-dissolve. 7) Resuspend the DM pellet in a small volume of TE or water. Low-speed vortexing or pipetting, with a dropper, while gradually increasing the TE, mixed until fully dissolved DNA, 1- 5χ 10 ⁶ cells per extracted plus about lul.

 The following steps 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.

8) Add RNase A to the DNA solution to a final concentration of 100ug / ml, and incubate at 37 ° C for 30 minutes. 9) Add SDS and proteinase K to the final concentration of 0.5¾ and 100ug / mL and 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 water phase, add 180 vols of 2mol / L sodium acetate and 2.5 vols of cold ethanol, and mix well--20 ° C for 1 hour. 13) Wash the pellet with 70% ethanol and 100% ethanol, air dry, and resuspend the nucleic acid. The process is the same as steps 3-6. 14) Measure A ₂₆ . And A ₂₈ . To check the purity and yield of DM. 15) Store at -20 ° C after dispensing.

 Preparation of sample film:

1) Take 4 x 2 pieces of nitrocellulose membrane (NC membrane) of appropriate size, and mark the spotting position and sample number on it with a pencil. Two NC membranes are required for each probe in order to follow the experimental steps. The film was washed with high-strength conditions and strength conditions, respectively. 2) Pipette and control 15 μl each, spot on the sample film, and dry at room temperature.

 3) Place on filter paper infiltrated with 0.1 mol / L NaOH, 1.5 mol / L NaCl for 5 minutes (twice), dry and place in 0.5 mol / L Tris-HCl (pH 7.0), 3 mol / L NaCl filter paper for 5 minutes (twice) and allowed to dry.

 4) Clamped in clean filter paper, wrapped in aluminum foil, and dried under vacuum at 60-80 ° C for 2 hours.

 Labeling of probes

1) 3μ1 Probe (0.1OD / Ιθμΐ), add 2μ1 Kinase buffer, 8-10 uCi γ- ³² P-dATP + 2U Kinase to make up to a final volume of 20μ1.

 2) Incubate at 37 ° C for 2 hours.

 3) Add 1/5 volume of bromophenol blue indicator (BPB X

 4) Pass Sephadex G-50 column.

5) Start to collect the first peak before ³² P-Probe washes out (can be monitored by Monitor).

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

 7) Monitor the amount of isotope with a liquid scintillator.

8) combining the first peak was collected after ³² P-Prob _e is prepared as required (the second peak to the free γ- ³² Ρ - dATP).

 Pre-hybridization

Put the sample film in a plastic bag, add 3-1 Omg pre-hybridization solution (10xDenhardt>s; 6xSSC, 0.1 mg / ml CT DM (calf thymus DNA)) _{. After} sealing the bag, shake at 68 ° C in a water bath 2 hours.

 Cross

 Cut a corner of the plastic bag, add the prepared probe, seal the bag, and shake at 42 ° C in water overnight. Wash film:

 High-intensity washing film:

 1) Take out the sample membrane of hybridization.

 2) 2xSSC, 0.1 SDS, 40. C wash for 15 minutes (twice).

 3) Wash in 0.1xSSC, 0.1% SDS for 15 minutes at 40 ° C (twice).

 4) 0.1xSSC, 0.1 ° / »SDS, 55. Wash for 30 minutes (twice) and dry at room temperature. Low-intensity washing film:

 1) Take out the hybridized sample membrane.

 2) 2xSSC, 0.1% SDS, 37. C Wash for 15 minutes (twice).

3) 0.1xSSC, 0.1% SDS, 37. C wash for 15 minutes (2 times). 4) Wash in 0.1xSSC, 0.1% SDS at 40 ° C for 15 minutes (twice), and dry at room temperature. X-ray auto-development:

 -70 ° C, X-ray autoradiography (pressing time depends on the radioactivity of the hybrid spot).

 Experimental results:

 The hybridization experiments performed under low-intensity membrane washing conditions showed no significant difference in the radioactive intensity of the above two probes. However, in the hybridization experiments performed under high-intensity membrane washing conditions, the radioactive intensity of probe 1 was significantly stronger than that of hybridization spots. The radioactive intensity of the hybridization spot of the other probe. Therefore, the presence and differential expression of the polynucleotide of the present invention in different tissues can be analyzed qualitatively and quantitatively with the probe 1. Example 7 DNA Microarray

 Gene chip or gene microarray (DM Microarray) is a new technology currently being developed by many national laboratories and large pharmaceutical companies. The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information. The polynucleotide of the present invention can be used as a target DM for gene chip technology for high-throughput research of new gene functions; searching for and screening new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases . The specific method steps have been reported in the literature. For example, see DeRi si, L L., Lyer, V. & Brown, P. 0 (1997) Science 278, 680-686. And Hel le, RA. , Schema, M., Chai, A., Shalom, D., (1997) PNAS 94: 2150-2155.

 (A) spotting

 A total of 4,000 polynucleotide sequences of various full-length cDNAs are used as the target MA, including the polynucleotide of the present invention. They were respectively amplified by PCR, and the concentration of the amplified product was adjusted to about 500ng / ul after purification. The spots were spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 μίη. The spotted slides were hydrated and dried, cross-linked in a UV cross-linker, and dried after elution to fix the DNA on the glass slides to prepare chips. The specific method steps have been reported in the literature. The sample post-processing steps in this embodiment are:

 1. Hydration in a humid environment for 4 hours;

 2. 0.2% SDS was washed for 1 minute;

3. Wash twice with ddH ₂ 0 for 1 minute each time;

4. NaBH _{4 is} blocked for 5 minutes;

5. 95 ° C water for 2 minutes; 6. 0 ° 2 ° /. Wash with SDS for 1 minute;

7. Rinse twice with ddH ₂ 0;

 8. Dry dry, 25. C Store in a dark place for future use.

 (Two) probe marking

 Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and the mRNA was purified using Oligotex raRNA Midi Kit (purchased from QiaGen). Cy3dUTP (5-Amino-propargyl-2'-deoxyuridine 5'-triphate coupled to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech) was used to label mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP (5-Amino-propargyl- 2 ' -deoxyuridine 5'-triphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech Company, labeled mRM, a specific tissue (or stimulated cell line) of the body, and purified the probe to prepare a probe. For specific steps and methods, see:

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

 (Three) cross

 The probes from the above two tissues and the chip were respectively hybridized in a UniHyb ™ Hybridizat ion Solut ion (purchased from TeleChem) hybridization solution for 16 hours, and washed with a washing solution (lx SSC, 0.2% SDS) at room temperature. Scanning was performed with a ScanArray 3000 scanner (purchased from General Scanning, USA), and the scanned images were analyzed and processed with Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.

 The above specific tissues (or stimulated cell lines) are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, Arsenic stimulated the L02 cell line and prostate tissue for 1 hour. Based on these 13 Cy3 / Cy5 ratios, draw a bar graph (Figure 1). It can be seen from the figure that the expression profiles of human RNA helicase 10 and human RNA helicase 95 according to the present invention are very similar.

Claims

Rights request

1. An isolated polypeptide-human RNA helicase 10, characterized in that it comprises: a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or an active fragment, analog or derivative thereof.

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

 3. The polypeptide according to claim 2, characterized in that it comprises a polypeptide having the amino acid sequence shown in SEQ ID NO: 2.

 4. An isolated polynucleotide, characterized in that said polynucleotide comprises one selected from the group consisting of:

 (a) a polynucleotide encoding a polypeptide having an amino acid sequence shown in SEQ ID NO: 2 or a fragment, analog, or derivative thereof;

 (b) a polynucleotide complementary to polynucleotide (a); or

 (c) a polynucleotide that is at least 70% identical to) or (b).

 5. The polynucleotide according to claim 4, wherein the polynucleotide comprises a polynucleotide encoding an amino acid sequence shown in SEQ ID NO: 2.

 6. The polynucleotide according to claim 4, characterized in that the sequence of the polynucleotide comprises the sequence of positions 738-1013 in SEQ ID NO: 1 or the sequence of positions 1-2421 in SEQ ID NO: 1. .

 7. A recombinant vector containing an exogenous polynucleotide, characterized in that it is a recombinant constructed from the polynucleotide according to any one of claims 4 to 6 and a plasmid, virus, or carrier expression vector Carrier.

 8. A genetically engineered host cell containing an exogenous polynucleotide, characterized in that it is a host cell selected from the group consisting of:

 (a) a host cell transformed or transduced with the recombinant vector of claim 7; or

 (b) a host cell transformed or transduced with a polynucleotide according to any one of claims 4-6.

 9. A method for preparing a polypeptide having human RM helicase 10 activity, characterized in that the method includes:

 (a) culturing the engineered host cell according to claim 8 under conditions that express human RNA helicase 10;

 (b) Isolating a polypeptide having human R helicase 10 activity from the culture.

10.An antibody capable of binding to a polypeptide, characterized in that the antibody is capable of binding to human RNA helicase 10 Specific binding antibodies.

 11. A class of compounds that mimic or regulate the activity or expression of a polypeptide, characterized in that they are compounds that mimic, promote, antagonize or inhibit the activity of human RNA helicase 10.

 12. The compound according to claim 11, characterized in that it is an antisense sequence of a polynucleotide sequence or a fragment thereof as shown in SEQ ID NO: 1.

 13. An application of the compound according to claim 11, characterized in that the compound is used for a method for regulating the activity of human RM helicase 10 in vivo and in vitro.

 14. A method for detecting a disease or susceptibility to a disease associated with a polypeptide according to any one of claims 1-3, characterized in that it comprises detecting the expression level of the polypeptide, or detecting the activity of the polypeptide Or detecting a nucleotide variation in a polynucleotide that causes abnormal expression or activity of the polypeptide.

 15. The use of the polypeptide according to any one of claims 1-3, which is characterized in that it is used for screening mimetics, agonists, antagonists or inhibitors of human RM helicase 10; or Identification of peptide fingerprints.

 16. The use of a nucleic acid molecule according to any one of claims 4-6, characterized in that it is used as a primer for a nucleic acid amplification reaction, or as a probe for a hybridization reaction, or for manufacturing a gene chip Or microarray.

 17. Use of a polypeptide, polynucleotide or compound according to any one of claims 1 to 6 and 11, characterized in that said polypeptide, polynucleotide or mimetic, agonist, antagonist The agent or inhibitor is composed of a safe and effective dose with a pharmaceutically acceptable carrier as a pharmaceutical composition for diagnosing or treating a disease associated with human RNA helicase 10 abnormality.

 18. The claim of any one of claims 1-6 and 11, the use of the polypeptide, polynucleotide or compound as described in claim 1, characterized in that the polypeptide, polynucleotide or compound is used for the preparation of a treatment such as malignant pimple. Tumors, blood diseases, HIV infections and immune diseases and drugs of various inflammations.