WO2001038377A1 - A novel polypeptide - human bromodomain-containing protein 95 and a polynucleotide encoding the same - Google Patents

Abstract

The present invention discloses a novel polypeptide - human bromodomain-containing protein 95 and a polynucleotide encoding the same, as well as a method of producing the polypeptide by DNA recombinant technology. The present invention also discloses methods of using the polypeptide in treatment of various disease, such as malignant tumor, blood disease, HIV infection, immunological diseas and various inflammation. The present invention also discloses and antagonist against the polypeptide and the therapeutic use of the same. The present invention also discloses the use of such polynucleotide encoding human bromodomain-containing protein 95.

Description

 A new polypeptide-a human bromodomain-containing protein 95 and a polynucleotide encoding the polypeptide TECHNICAL FIELD

 The present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, a human bromoyl group-containing protein 95, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique

 There are a class of proteins in the body, all of which contain bromodomains, and these proteins constitute an independent protein family. Members of this family are related to the correct binding of DNA and transcriptional regulation in the organism, especially the regulation of protein-protein interactions during the growth and development of the organism. They usually regulate the transcriptional expression of various proteins through the transcriptional regulation of downstream regulatory genes. Abnormal expression may lead to abnormal protein function, which leads to abnormal development of various parts of the body, triggering various related developmental disorders, immune disorders, and even various tumors and cancers.

 The bromodomain consists of the following conserved sequences:

 [STANVF] -X (2) -FX (4)-[DNS] -X (5, 7)-[DENQTF] — Y— [HFY] — X (2) _ [LIVMFY] -X (3)-[ LIVM] -X (4)-[LIVM] -1 (6, 8) -YX (12, 13)-[LIVM]-X (2)-N- [SACF]-X (2)-[FY].

 This domain is a structural motif composed of 77 amino acid residues. This structural motif also exists in various regulatory proteins in yeast and humans, and it constitutes a new regulatory protein family [Tamkim JW, Deuring R Et al., Cell, 1992, 68: 561-572]. This structural motif participates in protein-protein interactions in vivo, which may play an important role in regulating the activity and aggregation of multi-component complexes involved in transcriptional activity. If the domain or the protein containing the domain is abnormally expressed, it will cause some DNA transcriptional activity regulatory proteins to function abnormally, thereby affecting the expression of various proteins from the transcription level. This causes diseases associated with abnormal protein expression in various tissues and organs.

In 1997, Jones MH and others cloned a member of the family, RING3 protein, from humans. This protein is a mitogen-active protein that is expressed in proliferating cells including leukemia. This protein contains serine, threonine kinase, and an active site for spontaneous phosphorylation [Jones MH, Numata M et al., 1997, Genomics, 45: 529-534]. The study found that the gene has high homology with the developmental genes fsh and D26232 of Drosophila. These proteins all contain bromo-domain motifs and PEST sequences, and are specifically expressed in testis tissues. Therefore, these proteins play an important role in the development and regulation of organisms. Role.

 The new human bromodomain-containing protein of the present invention has 24% identity and 42% similarity at the protein level with the known human RING 3 'protein, and both similarities contain the bromodomain and PEST sequence fragments, so both are members of the same protein family and have similar biological functions. Abnormal expression may lead to abnormal protein function, resulting in abnormal development of various parts of the body, triggering various related developmental disorders, immune disorders, and even various tumors and cancers. As mentioned above, the human bromodomain-containing protein 95 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 more needs to be identified in the art Human bromodomain-containing protein 95 protein involved in these processes, and in particular the amino acid sequence of this protein was identified. The separation of the new bromine-containing protein 95 protein encoding 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 the disease, so it is important to isolate its coding DM. Disclosure of invention

 It is an object of the present invention to provide isolated novel polypeptides-human bromodomain-containing protein 95, as well as 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 bromo group-containing protein 95.

 It is another object of the present invention to provide a genetically engineered host cell containing a polynucleotide encoding a human bromo group-containing protein 95.

 Another object of the present invention is to provide a method for producing human bromodomain-containing protein 95.

 Another object of the present invention is to provide a human-brominated domain-containing protein directed to a polypeptide of the present invention.

95 antibodies.

 Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors directed to the human bromide-containing protein 95 of the polypeptide of the present invention.

 Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities of human bromodomain-containing protein 95.

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 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 120-2708 in SEQ ID NO: 1; and (b) a sequence having 1-2826 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 bromodomain-containing protein 95 protein, which comprises utilizing the polypeptide of the invention. The invention also relates to compounds obtained by this method.

 The present invention also relates to a method for in vitro detection of a disease or disease susceptibility associated with abnormal expression of human bromo group-containing protein 95 protein, comprising detecting mutations 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 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 for the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of human bromo group-containing protein 95.

 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: "Nucleic acid sequence" refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic 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 protein or polynucleotide "variant" refers to a protein or polynucleotide that has one or more amino acid or nucleotide changes Amino acid sequence or polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. Variants may have "conservative" changes in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine. Variants can also have non-conservative changes, such as replacing glycine with tryptophan.

 "Deletion" refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.

 "Insertion" or "addition" means that an alteration in the amino acid sequence or nucleotide sequence 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 "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 bound to a human bromodomain-containing protein 95, causes a change in the protein to regulate the activity of the protein. An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that binds a human bromodomain-containing protein 95.

 An "antagonist" or "inhibitor" refers to a molecule that blocks or regulates the biological or immunological activity of human bromodomain-containing protein 95 when combined with human bromodomain-containing protein 95. . Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that binds human bromodomain-containing protein 95.

 "Regulation" refers to a change in the function of human bromodomain-containing protein 95, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological properties and functions of human bromodomain-containing protein 95 Or changes in immune properties.

 "Substantially pure" means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify human bromodomain-containing protein 95 using standard protein purification techniques. The substantially pure human bromodomain-containing protein 95 produces a single main band on a non-reducing polyacrylamide gel. The purity of human bromodomain-containing protein 95 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 achieved by hybridization under conditions of reduced stringency (Southern Indian Traces or Northern blots). 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 a comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene software package, DNASTAR, Inc., Madison Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Cluster 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: The number of matching residues between sequence A and sequence B

X 100 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 the Cluster method or by methods known in the art such as Jotun Hein. J., (1990) Methods in emzumology 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 a "sense strand."

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

"Antibody" refers to a complete antibody molecule and its fragments, such as Fa,? (^ ') ₂ and? It can specifically bind to the epitope of human bromodomain-containing protein 95.

 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 matter from its original environment (for example, Natural environment). 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 in the present invention, "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 peptides in the natural state of living cells are not isolated and purified, but the same polynucleotides or peptides are separated and purified if they are separated from other substances existing in the natural state. .

 As used herein, "isolated human bromodomain-containing protein 95" refers to human bromodomain-containing protein 95 that is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify human bromodomain-containing protein 95 using standard protein purification techniques. Essentially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the human bromodomain-containing protein 95 polypeptide can be analyzed by amino acid sequence.

 The present invention provides a new polypeptide, a human bromodomain-containing protein 95, which basically consists of the amino acid sequence shown in SEQ ID NO: 2. The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide. The polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated, or may be non-glycosylated. The polypeptides of the invention may also include or exclude the initial methionine residue.

The invention also includes fragments, derivatives and analogs of human bromodomain-containing protein 95. As used herein, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially maintains the same biological function or activity of the human bromodomain-containing protein 95 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 (ΙΠ) Such a polypeptide sequence in which the mature polypeptide is fused with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV) a polypeptide sequence in which an additional amino acid sequence is fused into the mature polypeptide (Such as the leader sequence or secretory sequence or the sequence used to purify this polypeptide or protease sequence) As explained herein, such fragments, derivatives and analogs are considered to be skilled in the art. Within the knowledge of the staff.

 The present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2. The polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1. The polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 2826 bases in length and its open reading frame (120-2708) encodes 862 amino acids. According to the amino acid sequence homology comparison, it was found that this polypeptide has 28% homology with human RI NG 3 protein. It can be deduced that the human bromodomain-containing protein 95 has similar structure and function to human RI NG 3 protein. .

 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 in the present invention, but which differs from the coding region sequence shown in SEQ ID NO: 1.

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

 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 can 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 invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the 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) A denaturant was added during hybridization, such as 50% (v / v) formamide, 0.1 ° / »calf serum / 0.1% F i co ll, 42. C, etc .; or (3) hybridization occurs only when the identity between the two sequences is at least 95%, 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 herein, a "nucleic acid fragment" contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 cores. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques such as PCR to identify and / or isolate polynucleotides encoding human bromodomain-containing protein 95.

 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 of the present invention encoding human bromodomain-containing protein 95 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 DM 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 separation of cDM 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 mRNA extraction, and kits are also commercially available (Q i agene). And the construction of cDNA libraries is also a common method (Sambrook, et al., Molecule ar on, A Labora tory Manua, Collspring Harbor Labora tory. New York, 1989). Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.

 The genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (1) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of a marker gene function; (3) the determination of the level of human bromodomain-containing protein 95 transcripts (4) Detecting protein products expressed by genes 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 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 method (4), the protein product of the human bromodomain-containing protein 95 gene can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (EL I SA). Wait.

 A method using PCR technology to amplify DNA / RNA (Saiki, et al. Science 1985; 230: 1350-1354) 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 DM / RNA fragment 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 measured 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 the 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 bromodomain-containing protein 95 coding sequence, and the recombinant technology to produce the Polypeptide method.

 In the present invention, a polynucleotide sequence encoding a human bromo group-containing protein 95 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 expressed in bacteria (Rosenberg, et al. Gene, 1987, 56: 125); pMSXND expression vectors expressed in mammalian cells ( Lee and Nathans, J Bio Chem. 263: 3521, 1988) and 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, a marker gene, and translational regulatory elements.

Methods known to those skilled in the art can be used to construct expression vectors containing DM sequences encoding human bromo group-containing protein 95 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989). The DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: E. coli lac or trp promoter; Lambda phage PL promoter; eukaryotic promoter Promoters include the CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, retroviral LTRs, and other known controllable genes expressed in prokaryotic or eukaryotic cells or their viruses. Promoter. Expression vector also includes ribosomes for translation initiation Binding sites, transcription terminators, etc. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers 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 a human bromodomain-containing protein 95 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: E. 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 s melanoma cells, etc. .

Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art. 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 CaC l ₂ method used in steps well known in the art. The alternative is to use MgC l ₂ . 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.

 Using conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant human bromodomain-containing protein 95 (Scence, 1984; 224: 1431). Generally there are the following steps:

 (1) using the polynucleotide (or variant) of the present invention encoding human human bromodomain-containing protein 95, 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 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 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. 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 the amino acid sequence homology of the bromine group-containing protein 95 of the present invention and the human RING3 protein. The upper sequence is human bromodomain-containing protein 95, and the lower sequence is human RING3 protein. Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+".

 Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of human bromodomain-containing protein 95. 95kDa 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. In the following examples, the experimental methods without specific conditions are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer Suggested conditions.

 Example 1: Cloning of human bromodomain-containing protein 95

Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. Poly (A) mRM was isolated from total RNA using Quik mRNA Isolation Kit (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 multicloning site of pBSK (+) vector (Clontech) to transform DH5a. The bacteria formed a cDNA library. Dye terminate cycle react ion sequencing kit (Perk in-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer) were used to determine the sequences at the 5 'and 3' ends of all clones. Comparing the determined cDNA sequence with the existing public DNA sequence database (Genebank), it was found that the cDNA sequence of one of the clones 0735B09 was a new DM. A series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions. The results show that the 0735B09 clone contains a full-length cDNA of 2826bp (as shown in Seq ID N0: 1), with one from 120bp to 2708bp. The 2589bp open reading frame (ORF) encodes a new protein (as shown in Seq ID NO: 2). We named this clone PBS-0735B09, and the encoded protein was named human bromodomain-containing protein 95. Example 2: Homologous search of cDNA clones

 The sequence of the human bromodomain-containing protein 95 of the present invention and the protein sequence encoded by the protein were used by the Blast program (Basiclocal Alignment search tool) [Altschul, SF et a 1. J. Mol. Biol. 1990; 215: 403 -10], perform homology search in databases such as Genbank and Swiss sport. The gene with the highest homology to the human bromodomain-containing protein 95 of the present invention is a known human RING3 protein, and its accession number in Genbank is AF019085. The protein homology results are shown in Figure 1. The two are highly homologous, with an identity of 28% and a similarity of 42%. Example 3: Cloning of a gene encoding human bromodomain-containing protein 95 by RT-PCR

 CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer.

After purification of Qiagene's kit, PCR amplification was performed with the following primers:

 Primerl: 5,-GAAAACAAAAGAGATTGGCTGTGG —3, (SEQ ID NO: 3)

 Primer2: 5,-AATCAAATCATAACATTCCCTACT -3, (SEQ ID NO: 4)

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

 Primer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.

Amplification reaction conditions: 50 mmol / L l, 10 legs ol / L Tris-Cl, (pH8.5), 1.5 mmol / L MgCl ₂ , 200 Mmol / L dNTP, lOpmol primers in a 50 μ 1 reaction volume , 1U Taq DNA polymerase (Clontech). The reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94. C 30sec; 55 ° C 30sec; 72 ° C 2min. 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 linked to a pCR vector using a TA cloning kit (Unvitrogen product). The DNA sequence analysis results showed that the DM sequence of the PCR product was exactly the same as 1-2826bp shown in SEQ ID NO: 1. Example 4: Northern blot analysis of human bromodomain-containing protein 95 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 (pH 4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ) And 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. With 20μ _§ RNA, Electrophoresis was performed on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (ρίΠ.0)-5 mM sodium acetate-1 mM EDTA- ^2. 2M formaldehyde. It was then transferred to a nitrocellulose membrane. Preparation cc- ³² P dATP with ³² P- DNA probe labeled by the random primer method. The DNA probe used was the PCR-encoded human bromodomain-containing protein 95 coding region sequence (120bp to 2708bp) shown in FIG. 1. The 32P- labeled probe (approximately 2 X 10 ⁶ cpm / ml) and RNA was transferred to a nitrocellulose membrane overnight at 42 ° C in a hybridization solution, the solution comprising 50% formamide -25mMKH ₂ P0 ₄ ( pH7.4) -5 x SSC-5 x Denhardt's solution and 200 μg / ml salmon sperm DNA. After hybridization, the filters were washed in 1 X SSC-0.1% SDS at 55 ° (: 30rain. Then, analyzed and quantified using a Phosphor Imager. Example 5: In vitro expression of recombinant human bromodomain-containing protein 95 , Separation and purification

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

Primer3: 5'- CCCCATATGATGGGTGATGAGGTTTATTATTATTCC -3, (Seq ID No: 5) Primer4: 5,-CATGGATCCTTACCAACCAATTAAATTAGCCTTTG -3, (Seq ID No: 6) The 5 'ends of these two primers contain Ndel and BamHI obscure sites, respectively. The coding sequences of the 5 'and 3' ends of the gene of interest are followed, respectively. The Ndel and BamHI restriction sites correspond to the selectivity within the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Digestion site. The PCR reaction was performed using pBS-0735B09 plasmid containing the full-length target gene as a template. The PCR reaction conditions were as follows: a total volume of 50 μ1 containing 10 pg of pBS-0735B09 plasmid, primers Primer-3 and Primer-4 were lOpmol, 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 μg / ml), positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0735B09) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) by the calcium chloride method. In LB liquid medium containing kanamycin (final concentration 30 μg / ml), the host bacteria BL21 (pET-0735B09) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1 mmol / L, Continue incubation for 5 hours. The bacteria were collected by centrifugation, and the supernatant was collected by centrifugation, and the supernatant was collected by centrifugation. The affinity chromatography column His. Bind Quick Cartridge (product of Novagen) was used for chromatography to obtain 6 histidine (6His-Tag). The purified protein 95 containing human bromoyl domain was purified. After SDS-PAGE electrophoresis, a single band was obtained at 95 kDa (Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by the Edams hydrolysis method. As a result, the 15 amino acids at the N-terminus were completely identical to the 15 amino acid residues at the N-terminus shown in SEQ ID NO: 2. Example 6 Production of anti-human bromodomain-containing protein 95 antibodies

 A peptide synthesizer (product of PE company) was used to synthesize the following human bromodomain-containing protein 95-specific peptides:

NH ₂ -Met-G 1 y-Asp-G 1 u-Va 1-Tyr-Tyr-Phe-Arg-G 1 n- G l yH i sG l u- A 1 a-Tyr- COOH (SEQ ID NO: 7).

The polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively. For the method, see: Avrameas, et al. Immunochemi s try, 1969; 6: 43. Rabbits were immunized with 4 mg of the above-mentioned iL cyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost the immunity once. A 15 μg / ml bovine serum albumin peptide complex-coated titer plate was used as the ELI SA to determine the antibody titer in rabbit serum. Total I gG was isolated from antibody-positive rabbit serum using protein A-Sepharose. The peptide was bound to a cyanogen bromide-activated Se _P h _{ar 0} se4B column, and the anti-peptide antibody was separated from the total IgG by affinity chromatography. The immunoprecipitation method proved that the purified antibody could specifically bind to human bromodomain-containing protein 95. Industrial applicability

 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.

 Proteins containing specific bromo group domains such as RING3 and f sh play an important role in the transcriptional activity of homeoproteins. The bromodomain is involved in protein-protein interactions, and it may play an important role in regulating the activity and aggregation of multi-component complexes involved in transcriptional activity. Members of the bromodomain-containing protein family usually regulate the transcription and expression of various proteins through the transcriptional regulation of downstream regulatory genes, and regulate the interaction between proteins and proteins during the growth and development of organisms. Abnormal expression of these proteins can lead to abnormal protein function, which results in embryonic developmental disorders, children's growth disorders, various tumors and cancers, various inflammations, and immune disorders.

 It can be seen that the abnormal expression of the human bromodomain-containing protein of the present invention will produce various diseases, especially embryonic developmental disorders, children's growth disorders, various tumors and cancers, and various inflammations. These diseases include but are not Limited to:

Developmental disorders: cleft palate, facial cleft palate, cervical sac, cervical fistula, lack of limbs, limb differentiation disorders, gastrointestinal atresia or stenosis, ileal diverticulum, umbilical diaphragm, congenital umbilical hernia, congenital aganglion-free giant colon, Laryngeal tracheal stenosis or atresia, tracheoesophageal fistula, hyaline membrane disease, congenital pulmonary cyst, atelectasis, polyphrenic kidney, ectopic kidney, horse tellurium, double ureter, umbilical fistula, crypto, congenital inguinal hernia, double uterus , Vaginal atresia, suburethral Fissure, hermaphroditism, atrial septal defect, ventricular septal defect, abnormal arterial separation, aortic or pulmonary stenosis. Pulmonary stenosis, arterial duct occlusion, neural tube defect, congenital hydrocephalus, iris defect, congenital cataract, congenital Glaucoma or cataract, small eye deformity, congenital deafness, auricle deformity, Down syndrome, dwarfism of cartilage hypoplasia, spinal epiphyseal dysplasia, pseudochondral dysplasia, Lange rG i ed i on syndrome, funnel Thoracic, gonad hypoplasia, congenital adrenal hyperplasia, urethral fissure, short stature syndrome such as Conrad i syndrome and Danbolt-Clos s syndrome, congenital lens abnormality, congenital small eyelid Cleft palate, retinal dysplasia, congenital optic nerve atrophy, congenital sensorineural hearing loss, cleft foot and cleft foot, teratosis, Wi lli ams syndrome, Algille syndrome, Bayer syndrome, etc.

 Growth and development disorders: mental retardation, cerebral palsy, brain development disorders, familial cerebral nucleus dysplasia syndrome, skin, fat and muscular dysplasias such as congenital skin relaxation, premature aging, congenital horn Malformation, various metabolic defects such as various amino acid metabolic defects, stunting, dwarfism-sexual retardation, etc.

 Various inflammations such as allergic reactions, adult respiratory distress syndrome, pulmonary eosinophilia, rheumatoid arthritis, rheumatoid arthritis, osteoarthritis, cholecystitis, glomerulonephritis, dermatomyositis , Polymyositis, Addison's disease, telangiectasia, Bloom syndrome, xeroderma pigmentosum, etc.

 Carcinogenesis of various tissues: thyroid tumor, uterine fibroids, neuroblastoma, colon cancer, breast cancer, leukemia, lymphoma, malignant histiocytosis, melanoma, sarcoma, myeloma, teratoma, etc., adrenal cancer, Bladder cancer, bone cancer, bone marrow cancer, brain cancer, uterine cancer, gallbladder cancer, liver cancer, lung cancer, thymic tumor, etc.

 Abnormal expression of the human bromodomain-containing protein of the present invention will also cause certain hereditary, hematological and immune system diseases.

 The polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially the developmental disorders of embryos, children's growth disorders, various tumors and cancers, various Inflammation, some hereditary, hematological and immune system diseases. The invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human bromodomain-containing protein 95. Agonists enhance human bromodomain-containing protein 95 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, a mammalian cell or a membrane preparation expressing a human bromodomain-containing protein 95 can be cultured with a labeled human bromodomain-containing protein 95 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.

Antagonists of human bromodomain-containing protein 95 include selected antibodies, compounds, and receptor deficiencies Lost property and the like. Antagonists of human bromodomain-containing protein 95 can bind to human bromodomain-containing protein 95 and eliminate its function, 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 bromodomain-containing protein 95 can be added to bioanalytical assays by determining the effect of compounds on the interaction between human bromodomain-containing protein 95 and its receptors Determine if the compound is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds. Polypeptide molecules capable of binding to human bromodomain-containing protein 95 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, the 95 bromodomain-containing proteins should generally be labeled.

 The present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies. The invention also provides antibodies directed against a human bromodomain-containing protein 95 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.

 Polyclonal antibodies can be produced by injecting human bromodomain-containing protein 95 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 'S adjuvant and so on. Techniques for preparing monoclonal antibodies to human bromodomain-containing protein 95 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human Beta-cell hybridoma technology, EBV-hybridoma technology, etc. Inlay antibodies combining human constant regions and non-human-derived variable regions can be produced using existing techniques (Morr i son e t al, PNAS, 1985, 81: 6851). The existing technology for producing single chain antibodies (U.S. Pat No. 4946778) can also be used to produce single chain antibodies against human bromodomain-containing protein 95.

 Antibodies against human bromodomain-containing protein 95 can be used in immunohistochemical techniques to detect human bromodomain-containing protein 95 in biopsy specimens.

 Monoclonal antibodies that bind to human bromodomain-containing protein 95 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 bromodomain-containing protein 95 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 bromodomain-containing protein 95 Positive cells.

The antibodies of the present invention can be used to treat or prevent diseases related to human bromodomain-containing protein 95. Sick. Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human bromodomain-containing protein 95.

 The present invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human bromoyl-containing protein 95. These tests are well known in the art and include FI SH assays and radioimmunoassays. The level of human bromide-containing protein 95 detected in the test can be used to explain the importance of human bromide-containing protein 95 in various diseases and to diagnose human bromide-containing protein 95 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.

 Polynucleotides encoding human bromodomain-containing protein 95 can also be used for a variety of therapeutic purposes. Gene therapy techniques can be used to treat abnormal cell proliferation, development, or metabolism caused by the non-expression or abnormal / inactive expression of human bromodomain-containing protein 95. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human bromodomain-containing protein 95 to inhibit endogenous human bromodomain-containing protein 95 activity. For example, a mutated human bromodomain-containing protein 95 may be a shortened human bromodomain-containing protein 95 that lacks a signaling domain. Although it can bind to downstream substrates, it lacks signaling. active. Therefore, recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of human bromoyl-containing protein 95. Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding a human bromodomain-containing protein 95 into a cell. Methods for constructing recombinant viral vectors carrying a polynucleotide encoding a human bromodomain-containing protein 95 can be found in existing literature (Sambrook, et al.). In addition, a recombinant polynucleotide encoding human bromodomain-containing protein 95 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 bromodomain-containing protein 95 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 RNA and performs endonucleation. Antisense RNA, DNA, and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as the technology for the synthesis of oligonucleotides by solid-phase phosphoramidite chemical synthesis has been widely used. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RM. This DNA sequence has been integrated downstream of the vector's RNA polymerase promoter. To increase the stability of nucleic acid molecules, they can be modified in a variety of ways. For example, if the sequence length on both sides is increased, the linkage between ribonucleosides should use phosphorothioate or peptide bonds instead of phosphodiester bonds.

 Polynucleotides encoding human bromodomain-containing protein 95 can be used to diagnose diseases related to human bromodomain-containing protein 95. Polynucleotides encoding human bromo group-containing protein 95 can be used to detect the expression of human bromo group-containing protein 95 or abnormal expression of human bromo group-containing protein 95 in disease states. For example, the DNA sequence encoding human bromodomain-containing protein 95 can be used to hybridize biopsy specimens to determine the expression of human bromodomain-containing protein 95. Hybridization techniques include Sout hern blotting, Nor thern blotting, and in situ hybridization. These technical methods are all mature technologies that are publicly available, 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 (Microar ray) or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis of genes and genes in tissues. diagnosis. RNA-polymerase chain reaction (RT-PCR) in vitro amplification of human bromodomain-containing protein 95 specific primers can also detect human bromodomain-containing protein 95 transcripts.

 Detection of mutations in the human bromide-containing protein 95 gene can also be used to diagnose human bromide-containing protein 95-related diseases. Human bromodomain-containing protein 95 mutant forms include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human bromodomain-containing protein 95 DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR, and in situ hybridization. In addition, mutations may affect the expression of proteins. Therefore, Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.

 The sequences of the invention are also valuable for chromosome identification. 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 labeling 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 DM sequences on a chromosome.

 In short, PCR primers (preferably 15-35bp) can be prepared from cDNA to locate the sequence on the chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells that contain the human gene corresponding to the primer will produce amplified fragments.

 PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes. Using the oligonucleotide primers of the present invention, by a similar method, sublocalization can be achieved using a set of fragments from a specific chromosome or a large number of genomic clones. Sorted chromosomes are pre-screened and hybridized pre-selected to construct a chromosome-specific CDM library.

Fluorescent in situ hybridization (FI SH) of cDNA clones with metaphase chromosomes can be performed in one step Accurately locate chromosomes. For a review of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, 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, Mendelian Inheritance in Man (available online with Johns Hopkins University Welch Medical 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 cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals, and the mutation is observed in any normal individual, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable 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 bromodomain-containing protein 95 is administered in an amount effective to treat and / or prevent a specific indication. The amount and range of human bromodomain-containing protein 95 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. (1) General information:

 (ii) Title of invention: Human bromodomain-containing protein 95 and its coding sequence

(iii) Number of sequences: 7

(2) Information of SEQ ID NO: 1:

 (i) Sequence characteristics:

 (A) Length: 2826bp

 (B) Type: Nucleic acid

 (C) Chain: double strand

 (D) Topological structure: linear

 (ii) Molecular type: cDNA

(xi) Sequence description: SEQ ID NO: 1:

1201

1561 GGAGGAAGAAAAGAAACAGAAGCAGCTCTGTTTCCAGTAGTGCTGCATCAAGCCCTGAAA

2101 GAACCATTCAAGTAAATGGCCATGGAGGACAGCCATCAAAACTTGTGAAGAGGGGACCTG

2581 AGGATGACTCTGAAGAGGAGCAAAGGCAGCTGTTGTTCGAAGACACCTCTTTAACTTTTG

2821 TTGATT

(3) Information of SEQ ID NO: 2:

 (i) Sequence characteristics:

 (A) Length: 862 amino acids

(B) Type: Amino acid (D) Topological structure is linear

 (ii) Molecular type Polypeptide

 (xi) Sequence description SEQ ID NO :: 2:

 Met Gly Asp Glu Val Tyr Tyr Phe Arg Gin Gly His Glu Ala Tyr

Val Glu Met Ala Arg Lys Asn Lys lie Tyr Ser lie Asn Pro Lys

Lys Gin Pro Trp His Lys Met Glu Leu Arg Glu Gin Glu Leu Met

Lys lie Val Gly lie Lys Tyr Glu Val Gly Leu Pro Thr Leu Cys

Cys Leu Lys Leu Ala Phe Leu Asp Pro Asp Thr Gly Lys Leu Thr Gly Gly Ser Phe Thr Met Lys Tyr His Asp Met Pro Asp Val lie

Asp Phe Leu Val Leu Arg Gin Gin Phe Asp Asp Ala Lys Tyr Arg Arg Trp Asn lie Gly Asp Arg Phe Arg Ser Val lie Asp Asp Ala

Trp Trp Phe Gly Thr He Glu Ser Gin Glu Pro Leu Gin Pro Glu Tyr Pro Asp Ser Leu Phe Gin Cys Tyr Asn Val Cys Trp Asp Asn

Gly Asp Thr Glu Lys Met Ser Pro Trp Asp Met Glu Leu lie Pro

Asn Asn Ala Val Phe Pro Glu Glu Leu Gly Thr Ser Val Pro Leu

Thr Asp Gly Glu Cys Arg Ser Leu lie Tyr Lys Pro Leu Asp Gly

Glu Trp Gly Thr Asn Pro Arg Asp Glu Glu Cys Glu Arg lie Val

Ala Gly lie Asn Gin Leu Met Thr Leu Asp lie Ala Ser Ala Phe

Val Ala Pro Val Asp Leu Gin Ala Tyr Pro Met Tyr Cys Thr Val

Val Ala Tyr Pro Thr Asp Leu Ser Thr lie Lys Gin Arg Leu Glu

Asn Arg Phe Tyr Arg Arg Val Ser Ser Leu Met Trp Glu Val Arg

Tyr He Glu His Asn Thr Arg Thr Phe Asn Glu Pro Gly Ser Pro lie Val Lys Ser Ala Lys Phe Val Thr Asp Leu Leu Leu His Phe lie Lys Asp Gin Thr Cys Tyr Asn He lie Pro Leu Tyr Asn Ser

Met Lys Lys Lys Val Leu Ser Asp Ser Glu Asp Glu Glu Lys Asp

Ala Asp Val Pro Gly Thr Ser Thr Arg Lys Arg Lys Asp His Gin

Pro Arg Arg Arg Leu Arg Asn Arg Ala Gin Ser Tyr Asp lie Gin

Ala Trp Lys Lys Gin Cys Glu Glu Leu Leii Asn Leu lie Phe Gin Cys Glu Asp Ser Glu Pro Phe Arg Gin Pro Val Asp Leu Leu Glu

Tyr Pro Asp Tyr Arg Asp lie lie Asp Thr Pro Met Asp Phe Ala

Thr Val Arg Glu Thr Leu Glu Ala Gly Asn Tyr Glu Ser Pro Met

Glu Leu Cys Lys Asp Val Arg Leu He Phe Ser Asn Ser Lys Ala Tyr Thr Pro Ser Lys Arg Ser Arg lie Tyr Ser Met Ser Leu Arg 451 Leu Ser Ala Phe Phe Glu Glu His lie Ser Ser Val Leu Ser Asp

466 Tyr Lys Ser Ala Leu Arg Phe His Lys Arg Asn Thr lie Thr Lys

481 Arg Arg Lys Lys Arg Asn Arg Ser Ser Ser Val Ser Ser Ser Ala

496 Ala Ser Ser Pro Glu Arg Lys Lys Arg lie Leu Lys Pro Gin Leu

511 Lys Ser Glu Ser Ser Thr Ser Ala Phe Ser Thr Pro Thr Arg Ser

526 lie Pro Pro Arg His Asn Ala Ala Gin lie Asn Gly Lys Thr Glu

541 Ser Ser Ser Val Val Arg Thr Arg Ser Asn Arg Val Val Val Asp

556 Pro Val Val Thr Glu Gin Pro Ser Thr Ser Ser Ala Ala Lys Thr

571 Phe He Thr Lys Ala Asn Ala Ser Ala lie Pro Gly Lys Thr lie

586 Leu Glu Asn Ser Val Lys His Ser Lys Ala Leu Asn Thr Leu Ser

601 Ser Pro Gly Gin Ser Ser Phe Ser His Gly Thr Arg Asn Asn Ser

616 Ala Lys Glu Asn Met Glu Lys Glu Lys Pro Val Lys Arg Lys Met

631 Lys Ser Ser Val Leu Pro Lys Ala Ser Thr Leu Ser Lys Ser Ser

646 Ala Val He Glu Gin Gly Asp Cys Lys Asn Asn Ala Leu Val Pro

661 Gly Thr lie Gin Val Asn Gly His Gly Gly Gin Pro Ser Lys Leu

676 Val Lys Arg Gly Pro Gly Arg Lys Pro Lys Val Glu Val Asn Thr

691 Asn Ser Gly Glu lie lie His Lys Lys Arg Gly Arg Lys Pro Lys

706 Lys Leu Gin Tyr Ala Lys Pro Glu Asp Leu Glu Gin Asn Asn Val

721 His Pro lie Arg Asp Glu Val Leu Pro Ser Ser Thr Cys Asn Phe

736 Leu Ser Glu Thr Asn Asn Val Lys Glu Asp Leu Leu Gin Lys Lys

751 Asn Arg Gly Gly Arg Lys Pro Lys Arg Lys Met Lys Thr Gin Lys

766 Leu Asp Ala Asp Leu Leu Val Pro Ala Ser Val Lys Val Leu Arg

781 Arg Ser Asn Arg Lys Lys He Asp Asp Pro lie Asp Glu Glu Glu

796 Glu Phe Glu Glu Leu Lys Gly Ser Glu Pro His Met Arg Thr Arg

811 Asn Gin Gly Arg Arg Thr Ala Phe Tyr Asn Glu Asp Asp Ser Glu

826 Glu Glu Gin Arg Gin Leu Leu Phe Glu Asp Thr Ser Leu Thr Phe

841 Gly Thr Ser Ser Arg Gly Arg Val Arg Lys Leu Thr Glu Lys Ala

856 Lys Ala Asn Leu He Gly Trp

(4) Information of SEQ ID NO: 3

 (i) Sequence characteristics

(A) Length: 24 bases (B) Type: Nucleic acid

(C) Chain: single chain

 (D) Topological structure: linear

 (i i) Molecular type: Oligonucleotide

 (x i) Sequence description: SEQ ID NO: 3:

GAAAACAAAAGAGATTGGCTGTGG

(5) Information of SEQ ID NO: 4

 (i) Sequence characteristics

 (A) Length: 24 bases

 (B) Type: Nucleic acid

 (C) Chain: single chain

 (D) Topological structure: linear

 (Π) Molecular type: Oligonucleotide

 (x i) Sequence description: SEQ ID NO: 4:

AATCAAATCATAACATTCCCTACT

(6) Information of SEQ ID NO: 5

 (i) Sequence characteristics

 (A) Length: 32 bases

 (B) Type: Nucleic acid

 (C) Chain: single chain

 (D) Topological structure: linear

 (i i) Molecular type: Oligonucleotide

(x i) Sequence description: SEQ ID NO: 5: CAGCCATGGCGGGGAAGAAGAATGTTCTGTCG

(7) Information of SEQ ID NO: 6

 (i) Sequence characteristics

 (A) Length: 29 bases

 (B) Type: Nucleic acid

 (C) Chain: single chain

(D) Topological structure: linear (ii) Molecular type: Oligonucleotide

 (xi) Sequence description: SEQ ID NO: 6:

CCCGGATCCCGCTGCTTGGCCTTCTTCAC 29

(8) Information of SEQ ID NO: 7:

 (i) Sequence characteristics:

 (A) Length: 15 amino acids

 (B) Type: Amino acid

 (D) Topological structure: linear

 (ii) Molecular type: peptide

(xi) Sequence description: SEQ ID NO: 7:

 Met-Ala-Gly-Lys-Lys-Asn-Va 1-Leu-Ser-Ser-Leu-Ala-Va 1-Tyr-Ala 15

Claims

Claim

1. An isolated polypeptide-human bromodomain-containing protein 95, 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 an 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 the polynucleotide; 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 represented by SEQ ID NO: 2.

 6. The polynucleotide according to claim 4, characterized in that the sequence of the polynucleotide comprises the sequence of positions 120-2708 in SEQ ID NO: 1 or the sequence of positions 1-2826 in SEQ ID NO: 1. .

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

 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 a polynucleotide according to any one of claims 4-6.

9. A method for preparing a polypeptide having human bromo group-containing protein 95 activity, characterized in that the method comprises:

 (a) culturing the engineered host cell according to claim 8 under the condition that human bromodomain-containing protein 95 is expressed;

 (b) Isolating a polypeptide having human bromodomain-containing protein 95 activity 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 bromodomain-containing protein 95.

11. A class of compounds that mimic or regulate the activity or expression of a polypeptide, which are characterized in that they are compounds that mimic, promote, antagonize or inhibit the activity of human bromodomain-containing protein 95.

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 bromo group-containing protein 95 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. Use of a polypeptide according to any one of claims 1-3, characterized in that it is used for screening mimetics, agonists, antagonists or inhibitors of human bromodomain-containing protein 95; 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 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 1 1, characterized in that said polypeptide, polynucleotide or mimetic, agonist, antagonist The agent or inhibitor is composed of a safe and effective dose in combination with a pharmaceutically acceptable carrier as a pharmaceutical composition for diagnosing or treating a disease associated with a human bromodomain-containing protein 95 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. Disease, HI V infection and immune diseases and drugs of various inflammations.