WO2002048357A1 - A novel polypeptide, a human c0p9 compound subunit 37.62 and the polynucleotide encoding the polypeptide - Google Patents

Abstract

The present invention discloses a novel polypeptide, a human C0P9 compound subunit 37.62, 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 embryonic developmental deform, autoimmune disease, tumour diseases and researching in human antisenescence, etc. The invention also discloses the agonistes against the polypeptide and the therapeutic action thereof. The invention also discloses the uses of the polynucleotide encoding the novel human C0P9 compound subunit 37.62.

Description

 A new peptide one-to-human C0P9 complex subunit 37. 62

 And a polynucleotide encoding such a polypeptide

 The present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide—a human COP9 complex subunit 37.62, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and the polypeptide. Background technique

The COP9 (photomorphogenetic constituent 9) protein complex was first discovered in plants and is a protein complex composed of multiple subunits. It plays an important regulatory role in Arabidops is tha l iana light-mediated development. Recently, it has been found that a COP9 protein complex also exists in mammals [Ning Wei, Tomohiko Tsuge et al., Current Biology, 1998, 8: 919-922] ₀ People have obtained eight proteins that make up the mammalian COP9 protein complex Subunits and found that the COP9 complex is highly conserved in mammals, higher plants, and many eukaryotic multicellular organisms. All the subunits that make up COP9 are structurally homologous to the components of the transcriptional activator e lF3 and the proteasome regulatory complex. Therefore, they may have similar origins in evolution and similar biological functions in the living body. They are both important components of the multi-signal regulatory pathway in cells and regulate the normal differentiation, development, and expression of cells.

In mammals, the COP9 protein complex consists of eight subunits, namely SI-S8. Each subunit contains multiple different domains, which play different biological functions in the body. The S7 subunit is a recently discovered subunit and has no significant correlation with the proteasome. The S7 subunit has two proteins, S7a and S7b. These two proteins are 58% identical in mice and both contain a helix-helix structure, followed by a PCI domain [Ning We i, Tomohiko Tsuge et a l., Current Biology, 1998, 8: 919-922] ₀ Studies have found that this subunit may be part of the signal sequence of the COP9 protein complex, so it guides the correct localization of the complex in the cell in vivo The abnormal expression will cause the complex to fail to perform normal functions in the living body, thereby causing some diseases related to abnormal cell development, differentiation and expression. Studies have found that the COP9 protein complex is also involved in the process of signal transduction in humans, which may be related to the occurrence of multiple congenital abnormal metal retention syndrome (Smith-Magenis syndrome), whose abnormal expression will cause circadian rhythm Occurrence of disorders such as disorders, daytime narcolepsy, and nighttime insomnia [Lorra ine Potocki, Ken-Shiung Chen, et a l., Genomics, 1999, 57: 180-182] ₀ Gene chip analysis revealed that in the bladder mucosa, PMA + Ecv304 cell line, LPS + Ecv304 cell line thymus, normal fibroblasts 1024NC, Fibroblas t, growth factor stimulation, 1024NT, scar-forming fc growth factor stimulation, 1013HT, scar formation fc is not stimulated with growth factors, 1013HC, bladder cancer plant cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell lines, fetal skin, spleen, prostate cancer, jejunal adenocarcinoma, the expression profile of the polypeptide of the present invention is The expression profiles of the human COP9 complex subunits are very similar, so the functions of the two may also be similar. 62。 The present invention is named human COP9 complex subunit 37.62.

 As described above, the human COP9 complex subunit 37.62 protein plays an important role in regulating important functions of the body such as cell division and embryo development, and it is believed that a large number of proteins are involved in these regulatory processes, so more needs to be identified in the art The human COP9 complex subunit 37. 62 protein involved in these processes, in particular the amino acid sequence of this protein is identified. The newcomer C0P9 complex subunit 37. 62 The isolation of the protein-coding gene also provides a basis for research to determine the role of the protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding DNA. Disclosure of invention

 It is an object of the present invention to provide an isolated novel polypeptide-human COP9 complex subunit 37.62 and fragments, analogs and derivatives thereof.

 Another object of the invention is to provide a polynucleotide encoding the polypeptide.

 Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding the human COP9 complex subunit 37.62.

 Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human COP9 complex subunit 37.62.

 Another object of the present invention is to provide a method for producing human COP9 complex subunit 37.62.

 Another object of the present invention is to provide antibodies against the polypeptide-human COP9 complex subunit 37.62 of the polypeptide of the present invention.

 Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors directed to the polypeptide-to-human COP9 complex subunit 37.62 of the polypeptide of the present invention.

 Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of the human COP9 complex subunit 37.62.

The present invention relates to an isolated polypeptide, which is of human origin, and includes: 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 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 177-1205 in SEQ ID NO: 1; and (b) a sequence having 1-1624 in SEQ ID NO: 1 Sequence of bits.

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

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

 The invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit human COP9 complex subunit 37.62 protein activity, which comprises using the polypeptide of the invention. The invention also relates to compounds obtained by this method.

 The invention also relates to a method for in vitro detection of a disease or susceptibility to disease associated with abnormal expression of the human COP9 complex subunit 37.62 protein, comprising detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, Alternatively, the amount or biological activity of a polypeptide of the invention in a biological sample is detected.

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

 The present invention also relates to the preparation of a polypeptide and / or polynucleotide of the present invention for the treatment of {cancer, developmental disease or immune disease} or other drugs caused by abnormal expression of human COP9 complex subunit 37.62. use.

 Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein. The following terms used in this specification and claims have the following meanings unless specifically stated otherwise:

 "Nucleic acid sequence" refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and can also refer to genomic or synthetic DNA or RNA, which can be single-stranded or double-stranded, representing the sense strand or Antisense strand. Similarly, the term "amino acid sequence" refers to an oligopeptide, peptide, polypeptide or protein sequence and a fragment or part 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 protein or polynucleotide "variant" refers to an amino acid sequence having one or more amino acids or nucleotide changes, or a polynucleotide sequence encoding it. The change may include an amino acid sequence or an amino acid in a nucleotide sequence Or deletions, insertions or substitutions of nucleotides. Variants may have "conservative" changes in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine. Variants can also have non-conservative changes, such as replacing glycine with tryptophan.

 "Deletion" refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence. "Insertion" or "addition" refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule. "Replacement" refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.

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

 An "agonist" refers to a molecule that, when combined with human COP9 complex subunit 37.62, can cause the protein to change, thereby regulating the activity of the protein. An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to the human COP9 complex subunit 37.62.

 "Antagonist" or "inhibitor" refers to a molecule that can block or modulate the biological or immunological activity of human COP9 complex subunit 37.62 when combined with human COP9 complex subunit 37.62. . Antagonists and inhibitors can include proteins, nucleic acids, carbohydrates, or any other molecule that can bind to the human COP9 complex subunit 37.62.

 "Regulation" refers to a change in the function of human COP9 complex subunit 37. 62, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological properties and functions of human COP9 complex subunit 37. 62. Or changes in immune properties.

 "Substantially pure '" means essentially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify the human COP9 complex subunit 37. 62 using standard protein purification techniques. The substantially pure human COP9 complex subunit 37. 62 can produce a single main band on a non-reducing polyacrylamide gel. The human COP9 complex subunit 37. 62 The purity of the polypeptide can be analyzed by amino acid sequence.

 "Complementary" or "complementary" refers to the natural binding of a polynucleotide 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 can 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 hybridization can be inhibited by performing hybridization under conditions of reduced stringency (Southern or Northern blots, etc.) To detect. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean 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 the 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 (Higgins, D. G. and P. M. Sharp (1988) Gene 73: 237-244). The Clus ter method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula:

Number of matching residues between sequence A and sequence B

X 1 00 The number of residues in sequence A-the number of spacer residues in sequence A-the number of spacer residues in sequence B can also be determined by Clus ter method or by methods known in the art such as Jotun He in Percentage (He in J., (1990) Methods in emzumo logy 183: 625-645) ₀

 "Similarity" refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences. Amino acids used for conservative substitutions, 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 the "sense strand".

 "Derivative" refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.

"Antibody" refers to a complete antibody molecule and its fragments, such as Fa,? (') ₂ and? ^ It can specifically bind to the epitope of human COP9 complex subunit 37.62.

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 occurs naturally). For example, a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system. Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not a component of its natural environment, they are still isolated.

 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 existing in the natural state. .

 As used herein, "isolated human COP9 complex subunit 37. 62" refers to human COP9 complex subunit 37. 62 which is substantially free of other proteins, lipids, sugars, or other substances naturally associated with it. Those skilled in the art can purify the human COP9 complex subunit 37.62 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. Human COP9 complex subunit 37. 62 The purity of the peptide can be analyzed by amino acid sequence. '

 The present invention provides a new polypeptide one-to-one (: 0-9 complex subunit 37.62), which is basically composed of the amino acid sequence shown in SEQ ID NO: 2. The polypeptide of the present invention may be recombinant Polypeptides, natural polypeptides, synthetic polypeptides, and preferably recombinant polypeptides. The polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be obtained from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, Insect and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. The polypeptides of the present invention may also or may not include the starting formazan Methionine residue.

The invention also includes fragments, derivatives, and analogs of the human COP9 complex subunit 37.62. 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 COP9 complex subunit 37.62 of the present invention. A fragment, derivative, or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution The amino acid may or may not be encoded by a genetic codon; or (II) such a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or (III) such A type in which a mature polypeptide is fused to another compound (such as a compound that extends the half-life of a polypeptide, such as polyethylene glycol); or (IV) a type of polypeptide sequence in which an additional amino acid sequence is fused into a mature polypeptide ( Such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protease sequence) As explained herein, such fragments, derivatives and analogs are considered in It is within the knowledge of those skilled in the art.

 The present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2. The polynucleotide sequence of the present invention includes the nucleotide sequence of 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 1624 bases and its open reading frame of 177-1205 encodes 342 amino acids. According to the comparison of gene chip expression profiles, it was found that the polypeptide has a similar expression profile with the human COP9 complex subunit, and it can be inferred that the human COP9 complex subunit 37.62 has similar functions as the human COP9 complex subunit.

 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" refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.

 The invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention. Variants of this polynucleotide 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, 6 (TC; or (2) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Fi co ll, 42 ° C, etc .; or (3) only between two sequences Hybridization occurs when the identity is at least 95%, and more preferably 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 .

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 The acid is 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 COP9 complex subunit 37.62. 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 COP9 complex subunit 37. 62 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 the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature techniques for extracting mMA, and kits are also commercially available (Qiagene). Construction of cDNA libraries is also a common method (Sambrook, et al., Moleculolar Cloning, A Labora tory Manua, Coll Spring Harbor Labora tory. New York, 1989). Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When combined with polymerase reaction technology, 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): (D DNA-DNA or DNA-MA hybridization; (2) the presence or absence of a marker gene function; (3) determining the level of the transcript of the human COP9 complex subunit 37.62; (4) Detecting protein products of gene expression through immunological techniques or measuring biological activity. The above methods can be used alone 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 generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention. The genes or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).

 In the (4) method, the protein products expressed by the human COP9 complex subunit 37.62 gene can be detected using immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).

A method for amplifying DNA / RNA using PCR technology (Sa iki, eta l. Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention. Especially when it is difficult to obtain full-length cDNA from the library, With the RACE method (RACE-rapid cDNA end amplification method), primers for PCR can be appropriately selected based on the polynucleotide sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. The amplified DNA / RNA 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 DM fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequencing can also be performed 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 the polynucleotide of the present invention, and a host cell genetically engineered using the vector of the present invention or directly using the human COP9 complex subunit 37.62 coding sequence, and the recombinant technology to produce the described Polypeptide method.

 In the present invention, a polynucleotide sequence encoding the human COP9 complex subunit 37. 62 can be inserted into a vector to form a recombinant vector containing the polynucleotide of the present 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 suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, etal. Gene, 1987, 56: 125) expressed in bacteria; pMSXND expression vectors (Lee) expressed in mammalian cells and Nathans, J Bio Chem. 263: 3521, 1988) and baculovirus-derived vectors expressed in insect cells. In short, as long as it can be replicated and stabilized in the 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, marker genes, and translational regulatory elements.

 Methods well known to those skilled in the art can be used to construct a protein containing the human COP9 complex subunit 37.62.

An expression vector for the DNA sequence and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Labora tory Manua 1, cold harbor Harbor Labora tory. New York, 1989). The DNA sequence J'J can be operably linked to an appropriate promoter in an expression vector to guide mMA synthesis. Representative examples of these promoters are: the l ac 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, and the early and late SV40 promoters 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 and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs that act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancer with 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancer on the late side of the origin of replication, and adenovirus enhancer.

 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 regulatory elements (such as promoters, enhancers, etc.) and selectable marker genes.

In the present invention, a polynucleotide encoding human COP9 complex subunit 37.62 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a genetically engineered host containing the polynucleotide or the recombinant vector. cell. 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: Escherichia coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as fly S2 or Sf 9 _; animal cells such as CH0, COS or Bowes melanoma cells.

Transformation of a host cell with a 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 DNA uptake can be in the exponential growth phase were harvested, treated with (1 ₂ method used in the step are well known in the art. Alternatively, it is a MgCl _2. If If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging Wait.

 By conventional recombinant MA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant human COP9 complex subunit 37. 62 (Science, 1984; 224: 1431). Generally there are the following steps:

(1) using the polynucleotide (or variant) encoding the human-human COP9 complex subunit 37.62 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 media. 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 desired, recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional Refolding treatment, protein precipitant treatment (salting out method), centrifugation, osmotic lysis, 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. 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 chart of gene chip expression profiles of the present inventor's COP9 complex subunit 37.62 and human COP9 complex subunit. The upper graph is a graph of the expression profile of the human C0P9 complex subunit 37. 62, and the lower graph is the graph of the expression profile of the human C0P9 complex subunit. Among them, 1-fetal brain, 2-bladder mucosa, 3-PMA + Ecv304 cell line, 4-LPS + Ecv304 cell line thymus, 5-normal fibroblasts 1024NC, 6- Fibroblas t, growth factor stimulation, 1024NT, 7- Scar-like fc growth factor stimulation, 1013HT, 8-scar scar-fc stimulation without growth factor, 1013HC, 9-bladder cancer plant cells EJ, 10-bladder cancer, 11-bladder cancer, 12-liver cancer, 13-liver cancer cells Strain, 14-fetal skin, 15-spleen, 16-prostate cancer, 17-jejunum adenocarcinoma.

FIG. 2 is a polyacrylamide gel electrophoresis diagram of isolated human COP9 complex subunit 37. 62 (308-? Human 08). 10 ^) _& is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention

 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 without specific conditions in the following examples are generally based on conventional conditions such as Sambrook et al. Molecular cloning: Laboratory Manual (New York: Cold Spr ing Harbor

Laboratory Pres s, 1989), or as recommended by the manufacturer.

 Example 1: Cloning of human COP9 complex subunit 37.62

Human fetal brain total MA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. Poly (A) mRNA was isolated from total RNA using Quik mRNA Isolat ion Kit (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA. The Smart cDNA cloning kit (purchased from Clontech) was used to insert the cDNA fragment into the pBSK (+) vector (Clontech), and then transformed into DH5a to form a cDNA library. Dye terminate cycle react ion sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer) were used to determine the sequences at the 5 'and 3' ends of all clones. The determined CDM sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 1058el0 was new DNA. A series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions. The results showed that the 1058el0 clone contained The full-length cDNA is 1624bp (as shown in Seq ID NO: 1), and there is a 1029bp open reading frame (ORF) from 177bp to 1205bp, which encodes a new protein (as shown in Seq ID NO: 2). We named this clone pBS-lO ^ elO, and the encoded protein was named human COP9 complex subunit 37.62. Example 2: Cloning the human (0? 9 complex subunit) using the method! ^-? «1 37. 62 genes

 CDNA was synthesized using fetal brain cell total A as a template and ol igo-dT as a primer for reverse transcription reaction. After purification using Qiagene's kit, the following primers were used for PCR amplification:

 Pr imerl: 5 GGGAGGGATTGGGAGGCCCACGCC-3 '(SEQ ID NO: 3)

 Pr iraer2: 5-AAAGGTGACGAGTTTTTTAAATAA -3 '(SEQ ID NO: 4)

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

 Pr imer2 is the 3'-end reverse sequence in SEQ ID NO: 1.

Amplification reaction conditions: 50 m 1 of reaction volume containing ⁵⁰ mraol / L KC1, 10 ol / L Tris-CI, (pH 8. 5), 1.5 mmol / L MgCl ₂ , 200 μ mol / L dNTP, lOpmol primer, 1U Taq DNA polymerase (Clontech). On PE9600 DNA Thermal Cycler (Perkin- Elmer Corporation) according to the following reaction conditions are ^{25 cycles: 9 4 C 30sec; 55 °} C 30sec; 72 C 2min. During RT-PCR, β-act in was set as a positive control and template blank was set as a negative control. The amplified product was purified using a QIAGEN kit and ligated to a pCR vector (Invitrogen) using a TA cloning kit. DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as l-1624bp shown in SEQ ID NO: 1. Example 3: Northern blot analysis of human COP9 complex subunit 37.62 gene expression:

Total RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] ₀ This method involves acid guanidinium thiocyanate-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. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water. Using 20 g of RNA, perform electrophoresis on a 1.2% agarose gel containing 20 mM 3- (N-morphinyl) propanesulfonic acid (pH 7.0) ^-5 mM sodium acetate- ¹ mM EDTA-2.2 M formaldehyde . It was then transferred to a nitrocellulose membrane. ^{32 Ρ} dATP Preparation ³² P- DNA probe labeled by the random primer Method - with α. The DNA probe used was the PCR amplified human COP9 complex subunit 37.62 coding region sequence (177bp to 1205bp) shown in FIG. 1. 32P-labeled probes (approximately 2 10 ⁶ cpm / ml) were hybridized with a nitrocellulose membrane to which RNA was transferred at 42 C overnight in a solution containing 50% amidoamide-25 inM KH ₂ P0 ₄ (pH 7 4) -5 SSC-5 Denhardt's solution and 200 μg / ml salmon sperm DNA. After hybridization, the filter was washed in 1 SSC-0.1% SDS at 55 C for 30 min. Then, Phosphor Imager was used for analysis and quantification. Example 4: In vitro expression, isolation and purification of recombinant human COP9 complex subunit 37.62

 According to SEQ ID NO: 1 and the coding region sequence shown in FIG. 1, a pair of specific amplification primers was designed with the following sequence: Pr imer3: 5'-CATCCATGGATGATGTGCTCACGGGTGCCCTCT-3 '(Seq ID No: 5)

 Pr imer4: 5,-CATGGATCCCCTCTCCTTGGTTAGTGTCCTCTGG- 3, (Seq ID No: 6)

The 5 'ends of these two primers contain Ndel and BamHI restriction sites, respectively, followed by the coding sequences of the 5' and 3 'ends of the target gene, respectively. The Nde I and BamHI restriction sites correspond to the expression vector plasmid pET-28b (+) Selective endonuclease site on (Novagen, Cat. No. 69865. 3). The pBS-1058el 0 plasmid containing the full-length target gene was used as a template for the PCR reaction. The PCR reaction conditions were as follows: 10 pg of pBS-1058el0 plasmid, Primer-3, and Primer_4 were included in a total volume of 50 μ1; 10 pmol, Advantage polymerase Mix (Clontech) 1 μ1. 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. The ligation product was transformed into E. coli DH5a by the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 30 g / ml), positive clones were screened by colony PCR and sequenced. A positive clone (pET-1058el 0) with the correct sequence was selected, and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method. In LB liquid medium containing kanamycin (final concentration 30 μg / ml), the host strain 21 ( ₁ ^ 1 ¹ -1058610) was at 37. (: Cultivate to logarithmic growth phase, add IPTG to a final concentration of 1 mmol / L, and continue to cultivate for 5 hours. Centrifuge to collect the bacterial cells, ultrasonically break the bacteria, centrifuge to collect the supernatant, and use 6His-Tag ) Binding affinity column His. Bind Quick Cartr idge (Novagen) to perform chromatography to obtain the purified human protein COP9 complex subunit 37. 62. After SDS-PAGE electrophoresis, it was obtained at 1 OkDa. A single band (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, 15 amino acids at the N-terminus and the N-terminus shown in SEQ ID NO: 2 The 15 amino acid residues are identical. Example 5 Production of anti-human COP9 complex subunit 37.62 antibody

A peptide synthesizer (product of PE company) was used to synthesize the following human COP9 complex subunit 37. 62-specific peptides: NH2-Met-Met-Cys-Ser-Arg-Val-Pro-Ser-Glu-Gln-Ser- Ser-Gly-Thr-Ser-C00H (SEQ ID NO: 7). The peptide was coupled with hemocyanin and bovine serum albumin to form a complex. For the method, see: Avrameas, et ai. Immunochemi s try, 1969; 6: 43 ₀ 4 mg of the above hemocyanin peptide complex with complete Freund's adjuvant Rabbits were immunized, and 15 days later they were boosted with hemocyanin polypeptide complex and incomplete Freund's adjuvant. A titer plate coated with 15 μ _§牛 bovine serum albumin peptide complex was used as an ELISA to determine the antibody titer in rabbit serum. Total _Ig G was isolated from antibody-positive rabbit serum using protein A-Sepharose. The peptide was bound to a cyanogen bromide-activated Sepharose4B column, and the anti-peptide antibody was separated from the total IgG by affinity chromatography. Immunoprecipitation demonstrated purified antibody specifically binds to human C0P ⁹ complex subunit ^37.62. Example 6: Application of the polynucleotide fragment of the present invention as a hybridization probe

 Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways. For example, the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected. Further, the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissues or Whether the expression in tissue cells is abnormal.

 The purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method. 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 to hybridize the fixed polynucleotide sample to 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 sites of the sample on the filter are saturated with the carrier and the synthetic polymer. The pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid. After the hybridization step, the unhybridized probes are removed by a series of membrane washing steps. This embodiment uses 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 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 The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, then the primary probe should not be used;

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'-TGATGTGCTCACGGGTGCCCTCTGAACAGTCTTCTGGTACC-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'-TGATGTGCTCACGGGTGCCCCCTGAACAGTCTTCTGGTACC-3 '(SEQ ID NO: 9)

 Please refer to the literature for other commonly listed reagents and their preparation methods related to the following specific experimental procedures: DNA PROBES GHKeller; MMManak; Stockton Press, 1989 (USA) and more commonly used molecular cloning experiment manual books such as "Molecular Cloning" Experimental Guide (Second Edition, 1998) [US] Sambrook et al., Science Press.

 Sample Preparation:

1.Extract DNA from fresh or frozen tissue

Steps: 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 pellet (about 10 ml / g) with cold homogenization buffer (0.25 mol / L sucrose; 25 ol / L Tris-HCl, pH 7.5; 25 mmol / LnaCl; 25 ramol / L MgCl ₂ ). 4) Homogenize the tissue suspension at 4 ° C at full speed with an electric homogenizer until the tissue is completely broken. 5) Centrifuge at 1000g for 10 minutes. 6) Resuspend the cell pellet (1-5 ral per 0.1 gram of the initial tissue sample), and centrifuge at 1,000 g for 10 minutes. 7) Resuspend the pellet in lysis buffer (1 ml per 0.1 g of the initial tissue sample), and then follow the phenol extraction method below.

2, DNA phenol extraction method

Steps: 1) Wash cells with 1-10 ml of cold PBS and centrifuge at 1000 g for 10 minutes. 2) with cold cell lysate pelleted cells were resuspended (1 X 10 ⁸ cells / ml) Minimum application lOOul 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, 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. The DNA was then purified and ethanol precipitated.

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. Leave at -20C for 1 hour or overnight. 2) Centrifuge for 10 minutes. 3) Carefully aspirate or pour out the ethanol. 4) Use 70% Wash the pellet with 500ul of 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 evaporate the surface ethanol. Be careful not to allow the pellet to dry completely, otherwise it will be more difficult to re-dissolve. 7) Resuspend the DNA 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, the final concentration is 0.5% and 100ug / ml. 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 180 vols of 2mol / L sodium acetate and 2.5 vols of cold ethanol, mix and set at -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 3-5. 14) Determine A ₂₆ . And A ₂₈ . To detect the purity and yield of DNA. 15) Store at -20 ° C after dispensing.

Preparation of sample film:

 1) Take 4x2 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 needed for each probe, so that it can be used in the following experimental steps. The film was washed with high-strength conditions and strength conditions, respectively.

 2) Pipette and control 15 microliters each, spot on the sample film, and dry at room temperature.

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

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

 2) Incubate at 37 ° C for 2 hours.

 3) Add 1/5 volume of Bromophenol Blue Indicator (BPB).

 4) Pass Sephadex G-50 column.

5) Before the ³² P-Probe is washed out, start collecting the first peak (can be monitored by Monitor).

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

 7) Monitor the amount of isotope with a liquid scintillator

8) After the collection solution of the first peak is combined, the ³² P_Probe (the second peak is free γ- ³² P-dATP) to be prepared. Pre-hybridization

 Put the sample membrane in a plastic bag, add 3- l Omg pre-hybridization solution (l OxDenhardt's; 6xSSC, 0.1 mg / ml CT DNA (calf thymus should).), After sealing the bag, 68 ° C Shake in water bath for 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. P / oSDS, wash at 40 ° C for 15 minutes (twice).

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

 4) 0.1xSSC, 0.1 ° /. Wash in SDS at 55 ° C for 30 minutes (twice) and dry at room temperature.

 Low-intensity washing film:

 1) Take out the sample membrane of hybridization.

 2) 2xSSC, 0. Γ / oSDS, wash at 37 ° C for 15 minutes (twice).

 3) Wash in 0.1xSSC, 0.1% SDS at 37 ° C for 15 minutes (twice).

 4) Wash in 0.1xSSC, 0.1% SDS at 40 ° C for 15 minutes (twice), and dry at room temperature.

X-ray autoradiography:

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

 Experimental results:

 的 The hybridization experiments performed under low-intensity membrane washing conditions showed no significant difference in the radioactive intensity of the above two probe hybrid spots; while the hybridization experiments performed under high-intensity membrane washing conditions, the radioactive intensity of probe 1 was significantly stronger. To the radioactive intensity of the hybridization spot of another probe. Therefore, probe 1 can be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues.

Example 7 DNA Microarray

Gene microarrays or DNA microarrays are new technologies currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon, and then fluorescence 'detection and computer software are used to achieve the purpose of fast, efficient, and high-throughput analysis of biological information. The polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen 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, please refer to the literature DeRisi, JL, Lyer, V. & Brown, P.0.

 (1997) Science 278, 680- 686. and literature Helle, R. A., Schema, M., Chai, A., Shalom, D.,

(1 97) PNAS 94: 2150-2155.

 (A) spotting

 A total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were respectively amplified by PCR. After the purified amplified product was purified, the concentration was adjusted to about 500 ng / iil, and a Cartesian 7500 spotter (purchased from Cartesian, USA) was used to spot the glass medium. The distance is 280 μm. The spotted slides were hydrated, dried, and cross-linked in a UV cross-linker. After elution, the slides were fixed to fix the DNA on the glass slides to prepare chips. The specific method steps have been reported in the literature in various ways.

 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. Wash with 0.2% SDS for 1 minute;

7. Rinse twice with ddH ₂ 0;

 8. Dry and store at 25 ° C in the dark for future use.

 (Two) probe marking

 The total mRNA was extracted from the human mixed tissue and specific tissues (or stimulated cell lines) in one step, and the mRNA was purified by Oiigotex mRNA Midi Kit (purchased from QiaGen). Cy3dUTP (5-Amino-propargyl-2'-deoxyur idine 5'-triphate coupled to Cy3 fluorescent dye, purchased from Amersham Phamacia Biotech) was used to label the mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP (5-Amino-propargy 1- 2'-deoxyur idine 5'-triphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech, was used to label the mRNA of specific tissues (or stimulated cell lines) of the body, and the probe was prepared after purification. 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, R. W. (1995) Science. 270. (20): 467-480.

 (Three) cross

The probes from the two types of tissues and the chips were hybridized in a UniHyb ™ Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, and washed with a washing solution (1 x 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 fetal brain, bladder mucosa, PMA + Ecv304 cell line, LPS + Ecv304 cell line, thymus, normal fibroblasts 1024NC, Fibroblas t, growth factor stimulation, 1024NT, scar formation fc growth factor stimulation, 1013HT, scar into fc without growth factor stimulation, 1013HC, bladder cancer plant cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunal adenocarcinoma. Draw a graph based on these 18 Cy3 / Cy5 ratios. (figure 1 ) . It can be seen from the figure that the expression profile of the human COP9 complex subunit 37. 62 and the human COP9 complex subunit according to the present invention are very similar. Industrial applicability

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

 The human COP9 complex subunit 37. 62 and its derivatives of the present invention can also be used to treat diseases related to cell development and differentiation caused by abnormal expression of the human COP9 protein complex. These diseases include, but are not limited to, the following: Aldosterone excess, Addison's disease (ie, chronic adrenal insufficiency), Cushing's disease (ie, adrenal insufficiency), adrenal genital syndrome, some cancers (Including adenocarcinoma, leukemia, lymphoma, melanoma, sarcoma, etc.); specifically related to the following cancers: astrocytoma, neuroma, glioma, neuroblastoma, neuroblastoma, lung, esophagus, Cancer of the colon, bladder, kidney, liver, adrenal gland, prostate, penis, uterus, ovary and breast.

 The human COP9 complex subunit 37. 62 of the present invention can also be used to treat and prevent some inflammatory reactions caused by abnormal expression of the human COP9 protein complex. These inflammations include but are not limited to the following: allergic reactions, asthma, adult respiratory distress syndrome, rheumatoid arthritis, osteoarthritis, cholecystitis, glomerulonephritis, osteoporosis, dermatomyositis, and more Myositis, Addison's disease, Graves' disease, intestinal emergency syndrome, atrophic gastritis, erythematosus, myasthenia gravis, cerebral spinal cord multiple sclerosis, autoimmune thyroiditis, ulcerative colitis, anemia , Pancreatitis, segmental ileitis, myocarditis, periarterial sclerosis, multiple scleroderma, inflammation caused by infection and trauma, etc. .

The human COP9 complex subunit 37062 of the present invention may also be related to the occurrence of multiple congenital abnormal metal retention syndrome (Smith-Mageni s syndrome), whose abnormal expression will cause circadian rhythm disorders, daytime sleepiness, and nighttime insomnia And other diseases [Lorra ine Potocki, Ken-Shiung Chen, et al., Genomics, 1999, 57: 180-182]. The invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) the human COP9 complex subunit 37.62. Agonists enhance biological functions of the human COP9 complex subunit 37.62 to stimulate cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers. For example, a mammalian cell or a membrane preparation expressing a human COP9 complex subunit 37.62 can be cultured with a labeled human COP9 complex subunit 37.62 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.

 Antagonists of the human COP9 complex subunit 37.62 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human COP9 complex subunit 37. 62 can bind to and eliminate functions of human COP9 complex subunit 37. 62, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide to make the polypeptide Cannot perform biological functions.

 When screening compounds as antagonists, human COP9 complex subunit 37. 62 can be added to the bioanalytical assay by determining the effect of the compound on the interaction between human COP9 complex subunit 37. 62 and its receptors Determine if the compound is an antagonist. Receptor deletions and analogs that function as antagonists can be screened in the same manner as described above for screening compounds. Polypeptide molecules capable of binding to human COP9 complex subunit 37.62 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 37.62 molecules of the human COP9 complex subunit should be labeled.

 The present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen. These antibodies can be polyclonal or monoclonal antibodies. The invention also provides antibodies directed against the human COP9 complex subunit 37.62 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.

 Polyclonal antibodies can be produced by direct injection of human COP9 complex subunit 37.62 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 'S adjuvant and so on. Techniques for preparing monoclonal antibodies to human COP9 complex subunit 37.62 include, but are not limited to, hybridoma technology (ohler and Mistein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridization Tumor technology, EBV-hybridoma technology, etc. Chimeric antibodies that bind human constant regions and non-human-derived variable regions can be produced using existing techniques (Morrison et al., PNAS, 1985, 81: 6851). The unique technology for producing single chain antibodies (U.S. Pat No. 4946778) can also be used to produce single chain antibodies against the human COP9 complex subunit 37.62.

 Antibodies against the human C0P9 complex subunit 37. 62 can be used in immunohistochemical techniques to detect the human C0P9 complex subunit 37. 62 in biopsy specimens.

Monoclonal antibodies that bind to the human COP9 complex subunit 37. 62 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 It depends on the location of tumor cells and whether there is metastasis.

 Antibodies can also be used to design immunotoxins that target a particular part of the body. For example, human COP9 complex subunit 37. 62. High-affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.). A common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill the human COP9 complex subunit 37. 62 Positive cells.

 The antibodies of the present invention can be used to treat or prevent diseases related to the human COP9 complex subunit 37.62. Administration of an appropriate dose of antibody can stimulate or block the production or activity of human COP9 complex subunit 37.62.

 The invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human COP9 complex subunit 37.62. These tests are well known in the art and include FISH assays and radioimmunoassays. 62. The level of human COP9 complex subunit 37.62 detected in the test can be used to explain the importance of human COP9 complex subunit 37.62 in various diseases and to diagnose human COP9 complex subunit 37.62. A working disease.

 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 the human COP9 complex subunit 37. 62 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 C0P9 complex subunit 37.62. Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant human COP9 complex subunit 37.62 to inhibit endogenous human COP9 complex subunit 37.62 activity. For example, a variant human COP9 complex subunit 37. 62 may be a shortened human COP9 complex subunit 37. 62 that lacks a signaling domain, although it can bind to downstream substrates, but lacks signaling. active. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of the human COP9 complex subunit 37.62. Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding the human COP9 complex subunit 37.62 into a cell. A method for constructing a recombinant viral vector carrying a polynucleotide encoding the human COP9 complex subunit 37.62 can be found in the literature (Sambrook, et al.). In addition, the recombinant polynucleotide encoding human COP9 complex subunit 37. 62 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 COP9 complex subunit 37.62 mRNA are also within the scope of the present invention. A ribozyme is an enzyme-like RNA molecule that specifically breaks down a specific MA. The mechanism is that the ribozyme molecule specifically hybridizes with a complementary target RNA and performs endonucleation. Antisense RNA and DNA and ribozymes can be obtained by any existing MA or DNA synthesis technology, such as the technology of solid phase phosphate amide synthesis of oligonucleotides has been widely used. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA sequence is integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of a nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside linkages should use phosphate thioester or peptide bonds instead of phosphodiester bonds.

 The polynucleotide encoding the human COP9 complex subunit 37. 62 can be used for the diagnosis of diseases related to the human COP9 complex subunit 37. 62. The polynucleotide encoding the human C0P9 complex subunit 37. 62 can be used to detect the expression of the human C0P9 complex subunit 37. 62 or the abnormal expression of the human C0P9 complex subunit 37. 62 in a disease state. For example, the DNA sequence encoding the human C0P9 complex subunit 37. 62 can be used to hybridize biopsy specimens to determine the expression status of the human C0P9 complex subunit 37. 62. Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available. Some or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues. Human COP9 complex subunit 37.62 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect the transcription of human COP9 complex subunit 37.62.

 Detection of mutations in the human COP9 complex subunit 37.62 gene can also be used to diagnose human COP9 complex subunit 37.62-related diseases. Human COP9 complex subunit 37.62 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human COP9 complex subunit 37.62 DNA sequence. Mutations can be detected using well-known techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect the expression of proteins, so Northern blotting and Western blotting can be used to indirectly determine whether there is a mutation in the gene.

 The sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position of a human chromosome and can hybridize with 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) can be used to mark 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 mapped on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells containing human genes corresponding to the primers will produce amplified fragments.

PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes. Use this The oligonucleotide primer of the present invention can achieve sublocalization by a similar method using a set of fragments from a specific chromosome or a large number of genomic clones. Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct chromosome-specific cDNA libraries.

 Fluorescent in situ hybridization (FISH) of cDNA 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 Bas ic

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, for example, V. Mckusick, Mende l 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 individual, the mutation may be the cause of the disease. Comparing diseased and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable using cDNA sequence-based PCR. 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 contains a safe and effective amount of the polypeptide or antagonist, and carriers and excipients that 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 manufacture, use, or sell them. 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 COP9 complex subunit 37. 62 is administered in an amount effective to treat and / or prevent a specific indication. The amount and range of human COP9 complex subunit 37.62 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.

Claims

Request for Profit

1. An isolated polypeptide-human COP9 complex subunit 37.62, 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 of the polypeptide .

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, further comprising 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 the 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 (a) 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 177 to 1205 in SEQ ID NO: 1 or the sequence of positions 1-1624 in SEQ ID NO: 1. .

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

8. A genetically engineered host cell containing an exogenous polynucleotide, characterized in that it is selected from one of the following host cells:

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

 (b) a host cell transformed or transduced with any of the polynucleotides of claim 4-6.

9. A method for preparing a polypeptide having human COP9 complex subunit 37.62 activity, characterized in that the method includes:

 (a) culturing the engineered host cells described in claim 8 under the condition of expressing human COP9 complex subunit 37.62;

 (b) A polypeptide having human COP9 complex subunit 37.62 activity is isolated from the culture.

10. An antibody capable of binding to a polypeptide, characterized in that said antibody is an antibody capable of specifically binding to human COP9 complex subunit 37.62.

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 the human COP9 complex subunit 37.62.

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

13. Use of a compound according to claim 11, characterized in that the compound is used for a method for regulating the activity of human COP9 complex subunit 37.62 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 a polypeptide according to any one of claims 1-3, characterized in that it is used for screening a mimetic, agonist, antagonist or inhibitor of human COP9 complex subunit 37.62; or For 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 used to make a gene Chip or microarray.

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 mimetic, agonist, The antagonist or inhibitor is composed of a safe and effective dose with a pharmaceutically acceptable carrier as a pharmaceutical composition for the diagnosis or treatment of a disease associated with the human COP9 complex subunit 37.62 abnormality.

 18. The use of a polypeptide, polynucleotide or compound according to any one of claims 1-6 and 11, characterized in that the polypeptide, polynucleotide or compound is used for preparing for treating malignant tumors, blood, etc. Diseases, HIV infection and immune diseases and drugs of various inflammations.