WO2001023423A1

WO2001023423A1 - A novel gene encodes the human huntingtin interacting polypeptide which comprises the ww domain and its producing method and application

Info

Publication number: WO2001023423A1
Application number: PCT/CN2000/000274
Authority: WO
Inventors: Yumin Mao; Yi Xie
Original assignee: Shanghai Biorigin Gene Development Co. Ltd.
Priority date: 1999-09-14
Filing date: 2000-09-14
Publication date: 2001-04-05

Abstract

The invention disclosed a kind of human huntingtin interacting polypeptide which comprises the WW domain (WW-HIP). The polynucleotide encoding said polypeptide and a process for producing the polypeptide by recombinant methods. It also disclosed the method of applying the polypeptide for the treatment of various kinds of diseases, such as HD, immune disease, presbyophrenia, Duchenne muscular dystrophy, Liddle syndrome, DRPLA. The antibody, protagonist and antagonist of the polypeptide and their therapeutic usage for the above programmed cell death concerning diseases are also disclosed. In addition, the invention also disclosed the method to identify the mutation in the nucleic acid sequence of WW-HIP and the diagnostic method to examine the expression changes of the WW-HIP.

Description

Encoding a new human WW domain-containing Huntington protein binding

Polypeptide gene and application and preparation method thereof

The present invention belongs to the field of biotechnology, and in particular, the present invention relates to a polynucleotide sequence encoding a novel human WW domain-containing Huntington protein-binding polypeptide (WW-HIP) and a polypeptide encoded thereby. It also relates to the use of said polypeptides and polynucleotides in the diagnosis, prevention and treatment of diseases associated with abnormal expression of the protein. Background technique

The WW domain is composed of approximately 35 amino acid residues. Because it contains two very conserved tryptophan residues and one proline residue, it is named WW domain (WWWP AVWP domain) (Biochem.Biophys Res. Commun., 205: 1021-1205) (Trends Biochem. Sci. 19: 531-533 (1994)).

Both sides of the WW domain are rich in histidine or cysteine residues, such as dystrophin (Dystrophin), which suggests that it has the ability to bind metal ions (Nature Genetics 3, 283-291). The WW domain itself is in The four conserved aromatic amino acid residues are surrounded by a β-sheet structure. Both the hydrophobic core and a large number of charged residues indicate that this domain is a domain of protein-protein interaction. Although WW functional domains exist in proteins with different functions, they are involved in cell signaling and regulation.

Most of the WW functional domains interact with proline-rich domains and transmit signals (Proc.Natl.Acad.Sci.USA 92: 7819-7823 (1995). For example, the WW functional domains in FBP-11 and YAP65 are related to PPLP motif and PY motif binding of each ligand (J. Biol. Chem., 272, 17070-17077 (1997), (EMBO J., 16, 2376-2383

(1997)). A few WW domains depend on the phosphorylation pathway, the protein proline isomerase Pinl, The WW domain in the ubiquitin-binding enzyme Nedd4 binds to phosphoproteins. It has been confirmed that binding to phosphoserine or phosphothreonine is necessary for Pinl and its substrate to exert their physiological effects in vitro (Science, 1999 Feb 26, 283 (5406) 1325-1328).

Huntingtin contains a polyglutamine region, a proline-rich region, and 3-4 HEAT repeats. It is a protein necessary for embryonic development and nerve formation (Cell, 81, 811-823, 1995), (Nature Genet., 17,404-410). Prolongation of its mutant polyglutamine causes neurological disease Huntington's disease (HD) (Cell, 72, 971-983, 1993). Yeast two-hybrid experiments have found that Huntington protein only binds to various types of huntingtin-acting proteins (HYPs, HIPs) through its N-terminal enriched proline fragment, and the longer the neighboring polyglutamine fragment, the more its binding capacity Strong (Human Molecular Genetics, 1998, Vol. 7, No. 9 1463-1474)

Currently known HYP (HIP) has HYPA (murine FBP11 homologous protein) involved in protein splicing function (EMBO.J., 15,1045-1054, 1996), HYPF, HYPG (HIP2) involved in protein metabolism, and participate in specific Membrane functions: HYPI, HYPJ, HIP1 (Sla2p homologous protein of yeast cytoskeleton protein), CBS, and HYPB, HYPC, HYPD, etc. with no specific function. Among these proteins, HYPA, HYPB, and HYPC belong to the WW domain family. HYPA is found in the cytoplasm and nucleus. Northern blotting revealed that the mRNAs of three proteins are more abundant in adult and embryonic brains, suggesting that these proteins play different physiological functions in vivo after interacting with Huntington protein, thereby regulating the growth and development of the nervous system (Human Molecular Genetics, 1998, Vol. .7, No. 9 1463-1474 λ

As the WW domain-containing huntingtin-binding protein plays an important role in regulating the growth, development, and other important functions of the nervous system as described above, and it is believed that a large number of proteins are involved in these regulatory processes, more needs to be identified in the art WW domain-containing Huntington protein binding proteins involved in these processes, especially The amino acid sequence of this protein was identified. Isolation of a new WW domain-containing Huntington protein-binding protein encoding gene also provides a basis for determining 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. Summary of invention

In one aspect, the present invention provides an isolated WW domain-containing Huntington's protein-binding polypeptide, which comprises the amino acid sequence of SEQ ID No. 2, or a conservative variation polypeptide thereof, or an active fragment or derivative thereof.

In a preferred embodiment, the polypeptide of the invention is a polypeptide having the amino acid sequence of SEQ ID No. 2.

In another aspect, the invention provides an isolated polynucleotide, characterized in that it comprises a nucleotide sequence selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide as described above;

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

(c) A polynucleotide having at least 70% identity to a polynucleotide of (a) or (b). In a preferred embodiment, the polynucleotide of the present invention has

A polypeptide having the amino acid sequence shown in ID No. 2.

In another more preferred embodiment, the polynucleotide of the present invention is one selected from the group consisting of a sequence of positions 575 to 1849 in SEQ ID No. 1 and a sequence of positions 1-2507 in SEQ ID No. 1. the sequence of.

The present invention also relates to a recombinant vector containing the above polynucleotide and a host cell transformed, transfected or transduced with the polynucleotide or vector.

In yet another aspect, the present invention relates to a method for preparing a polypeptide having human WW functional domain Huntington protein binding polypeptide activity, which is characterized in that the method includes:

(a) Conditions suitable for expression of a WW domain-containing Huntington protein-binding polypeptide Culture the host cells; and

(b) Isolating a polypeptide having WW domain-containing Huntington's protein-binding polypeptide activity from the culture.

The invention also relates to an antibody that specifically binds to the WW domain-containing Huntington protein-binding polypeptide of the invention.

The present invention also relates to a method for screening compounds that mimic or regulate the activity or expression of a Huntington protein-binding polypeptide containing a WW functional domain, which is characterized by utilizing the polypeptide or polynucleotide of the present invention; and the simulation, promotion, Compounds that antagonize or inhibit the activity or expression of a polypeptide of the invention.

In addition, the present invention relates to a method for detecting a disease or disease susceptibility related to the polypeptide of the present invention, which is characterized by comprising:

(a) detecting an abnormal expression of the polypeptide;

(b) detecting abnormal activity of the polypeptide; or

(c) detecting a change in a nucleic acid associated with abnormal expression or activity of the polypeptide.

Finally, the present invention provides a pharmaceutical composition comprising an effective amount of a polypeptide of the present invention or a compound that mimics, promotes, antagonizes or inhibits the activity or expression of a polypeptide of the present invention, and a pharmaceutically acceptable carrier.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein. Detailed description of the invention

In one aspect, the present invention provides a substantially pure WW-HIP polypeptide consisting essentially of the amino acid sequence shown in SEQ ID No. 2. A feature of WW-HIP is that it has a WW domain consisting of about 35 amino acids. As used herein, "substantially pure" is that WW-HIP is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify WW-HIP using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reduced polypropylene amidamine gel. The purity of WW-HIP can be analyzed by amino acid sequence. The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.

The invention also includes fragments, derivatives and analogs of the polypeptide. 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 polypeptide described in the present invention. The polypeptide of the present invention includes a biologically active fragment, derivative, or analog of the polypeptide of SEQ ID No. 2, which may be compared to the polypeptide of SEQ ID No. 2 as follows: (I) wherein one or more amino acid residues are conserved or Non-conservative amino acid residues (preferably conservative amino acid residues) substituted and substituted amino acids may or may not be encoded by a genetic codon; or (II) wherein one or more amino acid residues include a substituent; or (III) Wherein the mature polypeptide is fused with another compound; or (IV) wherein the additional amino acid is fused into the mature polypeptide and used to purify the mature polypeptide. As set forth herein, such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art. In another embodiment, the present invention provides an isolated nucleic acid (polynucleotide) consisting essentially 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 2507 bases in length and its open reading frame encodes 424 amino acids. According to the amino acid sequence homology comparison, it was found that the polypeptide has 42% homology with human Huntingtin interating protein (HIP), and the polypeptide has a HIP gene functional region. It can be inferred that the new polypeptide has HIP similarity. Structure and function. 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. . 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 the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO. 1 or a degenerate variant. As used in the present invention, a "degenerate variant" refers to a nucleic acid sequence encoding a protein or peptide having SEQ ID NO2 but different from the coding region sequence shown in SEQ ID NO1 in the present invention. The polynucleotide encoding the mature polypeptide of SEQ ID NO. 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence. The term "polynucleotide encoding a polypeptide" is meant to include a polynucleoside encoding the polypeptide Acids and polynucleotides including 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 invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions. In the present invention, "stringent conditions" means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60C; or (2) addition of denaturation during hybridization Agents, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42C, etc .; or (3) the identity between the two sequences is at least 95%, and more preferably 97 Hybridization occurs only at% or more. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in FIG. 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 15 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 be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding WW-HIP. The invention also relates to a vector comprising a polynucleotide of the invention and a vector using the invention. A host cell produced by genetic engineering, and a method for producing a polypeptide according to the present invention by recombinant technology. The DNA sequence of the present invention can be obtained by several methods. For example, DNA is 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 nucleotide sequences, and 2) antibody screening of expression libraries to detect cloned DNA fragments that share structural characteristics .

The generation of specific DNA fragment sequences encoding WW-HIP can also be obtained by: 1) isolating double-stranded DNA sequences from genomic DNA; 2) chemically synthesizing DNA sequences to obtain double-stranded DNA of the desired polypeptide.

When the entire amino acid sequence of the desired polypeptide product is known, it can be obtained by direct chemical synthesis of the DNA sequence. If the entire sequence of the desired amino acid is unclear, the method of choice is the isolation of the cDNA sequence. The standard method for isolating cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature methods for extracting mRNA, and kits are also commercially available (Qiagene). Construction of cDNA libraries is also a common method (Sambrook, et al, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989). Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When combined with polymerized hydrazone reaction technology, even very few expression products can be cloned. These genes can be screened from these cDNA libraries by conventional methods. These methods include (but are not limited to): (1) DNA-DNA or DNA-RA hybridization; (2) the appearance or loss of function of a marker gene; (3) determination of the level of WW-HIP transcripts; (4) Detection of gene expression using immunological techniques or determination of biological activity Protein products. The above methods can be used singly or in combination.

In the method (1), the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 15 nucleotides, preferably 20-30 nucleotides, more preferably It is 50-60 nucleotides, preferably more than 100 nucleotides. The probe used here is generally a DNA sequence chemically synthesized based on the DNA sequence information of the gene of the present invention. The gene or a fragment of the present invention can of course be used as a probe. DNA probes can be labeled with radioisotopes, fluorescein, or enzymes (such as alkaline phosphatase).

In the (4) method, immunological techniques such as Western blots, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product of the WW-HIP gene expression. A method of using a PCR technique to amplify DNA / RNA (Saiki, et al. Science 1985; 230: 1350-1354) can be preferentially 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 in the above PCR can be appropriately based on the sequence information of the present invention disclosed herein. Select and synthesize using conventional methods. The amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis. The nucleotide sequence of the gene of the present invention obtained as described above, or various DNA fragments can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-54671) Acid sequencing can also be performed using commercial sequencing kits, etc. 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 before splicing into a full-length cDNA sequence. According to common recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce a recombinant WW-HIP polypeptide (Science, 1984; 224: 1431). Generally there are the following steps:

(1) transforming a suitable host cell with the polynucleotide (or variant) encoding the WW-HIP of the present invention or a recombinant expression vector containing the polynucleotide;

(2) host cells cultured in a suitable medium;

(3) Isolate and purify protein from culture medium or cells. In the present invention, the WW-HIP polynucleotide sequence can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors that are well known in the art. Vectors suitable for use in the present invention include, but are not limited to: T7-based expression vectors (Rosenberg, et al. 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, any plasmid and vector can be used as long as it can be replicated and stabilized in the host. An important feature of expression vectors is that they usually contain origins of replication, promoters, marker genes, and translation control elements. Methods known to those skilled in the art can be used to construct expression vectors for WW-HIP coding DNA sequences and suitable transcription / translation control signals. 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 laboraty. New York, 1989). The DNA sequence can be operably linked to an appropriate promoter in the expression vector to guide the mRNA binding to make. Representative examples of these promoters are: the lac or trp promoter of E. coli; the lambda phage PL promoter; eukaryotic promoters including CMV immediate early, HSV thymidine kinase, early and late SV40, retroviral LTRs and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. 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 for eukaryotic cell culture, neomycin resistance, and Green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli. Vectors containing the appropriate DNA sequences and appropriate promoters or control sequences described above can be used to transform appropriate host cells to enable them to express proteins. The host cell can be 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 of Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; animals of CHO, COS or Bowes melanoma cells Cells etc. When the polynucleotide of the present invention is expressed in higher eukaryotic cells, if an enhancer sequence is inserted into the vector, transcription will be enhanced. Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, that act on promoters to enhance gene transcription. Examples include 100 to 270 base pair SV40 enhancers on the late side of the origin of replication, and polyomas on the late side of the origin of replication Enhancers and adenovirus enhancers. Those of ordinary skill in the art will know how to select appropriate vectors, promoters, enhancers and host cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DNA from cells harvested after exponential growth phase, treated with <1 ₂ method used Buju well known in the art. Alternatively, MgCl ₂ is used. If necessary, transformation can also be performed by electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, and liposome packaging. The obtained transformants can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. 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. The recombinant polypeptide required in the above method is coated intracellularly, extracellularly, or expressed on the cell membrane or secreted extracellularly. 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. More specifically, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmosis, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography can be mentioned , Ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and these A combination of methods. Recombinant WW-HIP proteins or peptides have many uses. These uses include (but are not limited to) direct use as medications for diseases caused by WW-HIP hypofunction or loss, such as pseudohypertrophic malnutrition, Huntington's disease (HD), senile dementia, immune system disorders, Liddle syndrome DRPLA (dentatorubral and pallidoluysian atrophy), etc., and antibodies, peptides or other ligands used to screen or promote the function of WW domain-containing Huntington protein-binding polypeptides For example, antibodies can be used to activate or inhibit the function of WW-HIP. Screening peptide libraries with the expressed recombinant WW-HIP can be used to find therapeutically valuable polypeptide molecules that can inhibit or stimulate the function of WW-HIP.

The invention also provides methods for screening drugs to identify agents that increase (agonist) or suppress (antagonist) WW-HIP. For example, mammalian cells or membrane preparations expressing W-HIP can be cultured with labeled WW-HIP in the presence of drugs. The ability of the drug to increase or block this interaction is then determined.

Antagonists of WW-HIP include antibodies, compounds, receptor deletions, and analogs that have been screened. Antagonists of human WW-HIP can bind to human WW-HIP and eliminate its function, or inhibit the production of human WW-HIP, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions. Antagonists of human WW-HIP can be used to treat HD and immune system diseases.

When screening compounds as antagonists, this new human WW-HIP can be added to a bioanalytical assay to determine whether the compound is antagonistic by measuring the effects of this new human WW-HIP and its receptor Agent. In the same manner as the above-mentioned screening of compounds, it is possible to screen for receptor deletions and analogs that act as antagonists.

The polypeptide of the present invention can be used as a peptide 谙 analysis. For example, the polypeptide can be specifically cleaved by physical, chemical or enzyme, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis. Analysis.

A variety of methods can be used to produce antibodies against the WW-HIP 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.

Anti-WW-HIP antibodies can be used in immunohistochemistry to detect WW-HIR in biopsy specimens

Monoclonal antibodies that bind to WW-HIP 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.

The antibodies of the present invention can be used to treat or prevent WW-HIP-related diseases. Administration of appropriate doses of antibodies can stimulate or block the production or activity of WW-HIP.

Antibodies can also be used to design immunotoxins that target a particular part of the body. For example, WW-HIP 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 the 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 WW-HIP positive cells.

Polyclonal antibodies can be produced using WW-HIP or peptides to immunize animals, such as rabbits, mice, and rats. A variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant and the like.

WW-HIP monoclonal antibodies can be produced using hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497). 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 existing technology for producing single chain antibodies (U.S. Pat No. 4946778) can also be used to produce single chain antibodies against W-HIP.

Polypeptide molecules capable of binding to gastric-HIP can be obtained by selecting a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, you must Label WW-HIP molecules. The polypeptide of the present invention can be used in combination with a suitable medicinal plant. This composition comprises a therapeutically effective amount of a polypeptide, and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, glucose, water, glycol, ethanol, and combinations thereof. These formulations should be suitable for the mode of administration. The present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention. Along with these containers, there may be prompts in the form of instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which reminders permit their administration on the human body by government agencies that produce, 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 topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route. WW-HIP is administered in an amount effective to treat and / or prevent a specific indication. The amount and range of WW-HIP administered to a learner will depend on many factors, such as the mode of administration, the natural conditions of the person to be treated, and the judgment of the diagnostician. The invention also relates to a diagnostic test method for quantitative and localized detection of WW-HIP levels. These tests are well known in the art and include FLISH assays and radioimmunoassays. The level of WW-HIP detected in the test can be used to explain the importance of WW-HIP in various diseases and to diagnose diseases in which WW-HIP can work.

WW-HIP polynucleotides can be used for the diagnosis and treatment of WW-HIP related diseases Treatment. For diagnostic purposes, WW-HIP polynucleotides can be used to detect WW-HIP expression or abnormal expression of WW-HIP in disease states. For example, the DNA sequence of WW-HIP can be used to hybridize biopsy specimens to determine the abnormal expression of WW-HIP. Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and so on. These techniques and methods are publicly available and mature, and related kits are commercially available. A part or all of the polynucleotide of the present invention can be used as a probe to be fixed on a microarray (Microarray) or a DNA chip (DNA Chip) for analyzing differential expression analysis and gene diagnosis of genes in a tissue. WW-HIP transcription products can also be detected by in vitro amplification of RNA-polymerized alcohol chain reaction (RT-PCR) using primers specific to the WW domain-containing Huntington protein-binding polypeptide. Detection of mutations in the WW-HIP gene can also be used to diagnose WW-HIP-related diseases. The forms of WW-HIP mutations include point mutations, translocations, deletions, recombination and any other abnormalities compared to the DNA sequence of normal wild-type WW-HIP. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so Northern blots and Western blots can be used to indirectly determine whether a gene is mutated. The sequences of the invention are also valuable for chromosome identification. This sequence specifically targets a specific position of a single human chromosome and can hybridize to this chromosome. Moreover, there is currently a need to identify specific sites on the chromosome. Nowadays, few chromosome labeling reagents based on actual sequence data (repeat polymorphisms) are available for labeling chromosome positions. According to the present invention, the action of DNA on chromosomes is an important first step in correlating these sequences with the underlying solids associated with a disease.

Simply put, by preparing PCR primers (preferably 15-25bp) from cDNA, the sequence can be mapped to chromosomes. Quickly choose not to use computer analysis of cDNA Primers that span an exon of genomic DNA complicate the amplification procedure. These primers were then used for PCR screening of somatic hybrid cells containing a single human chromosome. Only these hybrid cells containing human genes corresponding to the primers will produce amplified fragments.

PCR mapping of somatic hybrid cells is a quick way to assign specific DNA to specific chromosomes. Using the present invention with the same oligonucleotide primers, a large number of genomic clone collections can be obtained from a group of experimental objects from a specific chromosome fragment or a similar manner. It can be similarly used for the color of the day Other strategies for somatic action include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-screening by hybridization to construct chromosomal specific cDNA libraries.

Fluorescent in situ hybridization (FISH) of cDNA clones to spread metaphase chromosomes can be used for precise chromosomal localization in one step. 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 mapped to 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, MendeIian Inheritance in Man I (available online with the Johns Hopkins University Welch Medical Library). Then, through association analysis, the relationship between genes and diseases that have been mapped to some chromosomal regions is determined.

Next, the difference in c D N A or genomic sequence between the affected and unaffected individuals needs to be determined. If mutations are observed in some or all diseased individuals, but not in any normal individuals, mutations may be the cause of the disease.

Using the resolution capabilities of current physical mapping and gene mapping techniques, the cDNA pinpointed 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 every 20kb—genes). Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible from chromosomal extension or detectable by cDNA sequence-based PCR. Finally, the complete sequencing of the genes of several individuals is needed to confirm the existence of mutations and the differences between mutations and polytypes.

WW-HIP polynucleotides 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 WW-HIP polypeptides without expression or abnormal / inactive WW-HIP polypeptide expression. Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant WW-HIP polypeptides and be used to inhibit endogenous WW-HIP polypeptide activity. For example, a variant WW-HIP polypeptide may be a shortened WW-HIP polypeptide lacking a signaling domain, and although it can bind to a downstream substrate, it lacks signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of WW-HIP polypeptide. Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer the WW-HIP polypeptide gene into cells. A method for constructing a recombinant viral vector carrying a WW-HIP polypeptide gene can be found in the existing literature (Sambrook et al.). In addition, the recombinant WW-HIP polypeptide gene can be packaged into liposomes and transferred into cells. Oligonucleotides (including antisense RNA and DNA) and nuclear cymbals that inhibit WW-HIP polypeptide mRNA are also within the scope of the present invention. Ribo alcohol 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 technology for synthesizing RNA or DNA. For example, the technology for the synthesis of oligonucleotides by solid-phase ammonium acetonide 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 RNA. This This DNA sequence has been integrated downstream of the RNA polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside linkage should use a phosphorothioate or peptide bond instead of a phosphonate diester bond.

Methods for introducing a polynucleotide into a tissue or cell include: injecting the polynucleotide directly into a tissue in vivo; or introducing the polynucleotide into a cell via a vector (such as a virus, phage, or plasmid) in vitro Then, the cells are transplanted into the body. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Sequence comparison of the WW-HIP peptide sequence (bottom row) with human Huntington's protein (HIP) (top row). The comparison length is 178 amino acids.

Score = 971 (341.8 bits), expectation = 1.8e-98, P = 1.8e-98

Identity = 178/178 (100%), Similarity = 178/178 (100%), frame

= +2. Figure 2: Photo of the results of the yeast two-hybrid experiment:

The semicircle on the medium is the result of (pLexA-HDl-425Q62) and (pB42AD-WW- HIP) transferred to yeast EGY48 with LEU2 and LacZ genes on the selective plate (SD-UraHisTrpLeu).

The lower half of the medium is the result of (pLexA-HDl-425Q62) and (pB42AD) co-transformed into yeast EGY48 strains with LEU2 and LacZ genes, and the results were obtained on a selective plate (SD-UraHisTrpLeu). The following examples will further illustrate the invention, but are not intended to limit the invention.

Example 1: Cloning of WW-HIP polypeptide cDNA Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. Poly (A) mRNA was isolated from total RNA using Quik mRNA Isolation Kit (Qiegene). 2ug poly (A) mRNA was 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 pUC118 and transformed into DH5ct bacteria to form a cDNA library. A total of 3028 clones were obtained. The dideoxy method was used to determine the sequences at the 5 'and 3' ends of all clones. The determined cDNA sequence was compared with the existing public DNA sequence database, and it was found that the DNA sequence of one clone 0273H12 was a new DNA. The DNA sequence contained in this clone was determined in both directions by synthesizing a series of primers. Computer analysis showed that the full-length cDNA was a new DNA sequence (SEQ ID NO1), with a 1275bp ORF from 575bp to 1849bp, encoding a new protein (SEQ ID NO2). We named this protein as the WW domain-containing Huntington protein-binding polypeptide (WW-HIP). Example 2: Cloning of WW-HIP polypeptide by RT-PCR

CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer for reverse transcription. After purification with Qiagen's kit, PCR was performed using the following primers:

Primer 1: 5, -TACCTACATCTGAACCAGAAGC-3, located at the beginning of SEQ ID No. 1-23bp;

Primer 2: 5'-GTTCTTTAATTGATTTTATTTT-3 'is located at 2485-2507bp of SEQ ID No.1.

Amplification reaction conditions: 50 mmol / L KCl, 10 mmol / L Tris-Cl, (pH 8.5), 1.5 mmol / L MgCl 2,200 μπιοΙ / L dNTP, 25 pmol primer, 2.5 U Taq DNA in a 50 μ1 reaction volume Polymerase. Reaction on a PE9600 DNA thermal cycler for 25 cycles under the following conditions: 94C 30sec; 55C, 30sec; 72C 2min. The template blank was set as a negative control during RT-PCR. After the amplified product QIAGEN kit was purified, it was ligated to a pCR vector (Invitrogen) using a TA cloning kit, and the DNA sequence was determined. As a result, the DNA sequence of the PCR product was exactly the same as that of 1-2507bp in SEQ ID No.1. Example 3: In vitro expression, isolation and purification of recombinant WW-HIP polypeptide

A pair of primers were designed at the start codon and the stop codon of the WW-HIP polypeptide gene.

Primer 3: 5'TCTAGAAGGCTCAGAAACAACA-3 '

Primer 4: 5'-AAGCTTCCAACAGTCACTCTAA-3 '

Its 5 'ends are respectively provided with Xbal and Hindlll restriction sites. WW-HIP polypeptide gene coding region was obtained by PCR amplification using plasmid 0273hl2 containing the full-length target gene as a template. After digestion, the amplified fragment was inserted into the expression vector pGEM-3Z (purchased from Pharmacia Biotech) and transformed into BL21 (DE3) pLysE. On the LB plate containing ampicillin and IPTG, five white recombinant transformants were screened for DNA sequence. As a result of analysis, the sequence of the coding region of the gene obtained in Example 1 was completely the same.

Pick a loop of recombinant bacteria and inoculate 20ml LB medium (containing ampicillin

100ug / ml), 37 "shake culture overnight as the seed liquid, take the seed liquid and transfer it to 4 liters of LB medium at 2% inoculation volume, 37t shake culture until the cell A ₆ 00 = 0.7 (logarithmic growth phase), IPTG was added to a final concentration of 0.4 mmoI / L, and the cells were cultured for another 12 hours. The cells were collected by centrifugation, washed with lxPBS with buffer A (16mM Na2HP04, 4mM NaH2P04, pH 6.5) at 10ml / g cells, and then dissolved in an ice bath. The supernatant was collected by sonication, centrifugation, and the supernatant was passed through a glutathione-Sepharose 4B affinity chromatography column. After washing with buffer A, elution was performed with an eluate containing 5 mM reduced glutathione. To obtain purified WW-HIP polypeptide. Example 4: Production of anti-WW-HIP polypeptide antibodies

A peptide synthesizer (PE-ABI) was used to synthesize WW-HIP peptide-specific peptides: NH ₂ -Gly-Tyr-Asn-Ala-Pro-His-His-Pro-Phe-Ala-Gly-Tyr-Pro- Pro- Gly-COOH. The peptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively. For methods, see: Avrameas. Immunochemistry, 1969; 6:43. Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex and complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex and incomplete Freund's adjuvant were used to boost immunity once. A titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum. Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose. The peptide was bound to a cyanogen bromide-activated Sepharose 4B column, and anti-peptide antibodies were separated from the total ¾G by affinity chromatography. The immunoprecipitation method proved that the purified antibody could specifically bind to WW-HIP polypeptide. Example 5: Homologous search of cDNA clones

The sequence of the polynucleotide of the polypeptide provided by the present invention and the protein sequence encoded by the polypeptide are used to perform homology search in databases such as Genbank, swissport, and the program for searching is called Blast (Basic local alignment search tool) (1993 Proc Nat Acasd Sci 90 : 5873-5877), Blast can find many genes that are homologous to the new human WW-HIP polypeptide. Among them, the gene with the highest homology with the gene we found is encoded by Genbank with accession number AF049103. These retrieved gene and protein sequences can be retrieved from Genbank. The recalled sequences can be compared using the Pileup (multi-sequence) and Gap (two-sequence) programs in the GCG software package. Functional prediction of new proteins can be analyzed using the Motif program. The results of the homology search are shown in Fig. 1 below. The results show that the human WW-HIP polypeptide provided by the present invention is identical with HIP in 178/178 (100%) and similarity in 178/178 (100%). This protein is a secreted protein. Example 6: Northern blot

Total R A was extracted in one step [Anal. Biochem 1987, 162, 156-159]. This method involves acid guanidinium thiocyanate benzopan-chloroform extraction. That is, 4M guanidinium isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0) were used to homogenize the tissue, and 1 volume of benzylphenol 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 mg of RNA, electrophoresis was performed on a 1.2% agarose gel containing 20 mM 3- (N-morphinyl) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-ImM EDTA-2.2M formaldehyde. It was then transferred to a nitrocellulose membrane. Hybridize with 32P-labeled probe (approximately 1 '106 cpm / ml) at 42 "overnight in a solution containing 50% formamide-25mM KH2P04 (pH7.4) -5' SSC-5 'Denhardt's solution and 200mg / ml salmon sperm DNA. A-32P dATP was used to prepare a 32P-labeled DNA probe by random primers. The DNA probe used was the WW-HIP polypeptide coding region sequence amplified by PCR. After hybridization, the filter was placed on Wash in 1 'SSC-0.1% SDS for 30 min. Then, analyze and quantify with Phosphor Imager.

The results showed that the WW-HIP peptide gene was mainly expressed in brain tissue and testis tissue. Example 7, yeast two-hybrid experiment

Using multiple cloning sites, the plasmid pLexA was fused to the N-terminus of the HD gene (pLexA-HDl-425Q62) and the plasmid pB42AD was fused to the WW-HIP gene (B42AD-WW-HIP) to be transferred into yeast EGY48 strains with LEU2 and LacZ genes. And then expressed on a selective plate (SD-UraHisTrpLeu) simultaneously with the negative control * (Fig. 2), and observe the expression of LEU2 and LacZ genes It can be judged whether Huntington protein and WW-HIP have an interaction relationship.

* Negative control is EGY48 strain transferred by pLexA-HDl-425Q62 and pB42AD

* Plasmids and bacteria used in two-hybrid experiments were purchased from Clontech

The results indicate that Huntington protein and WW-HIP can interact.

Claims

Rights request

What is claimed is: 1. An isolated WW domain-containing Huntington protein binding polypeptide comprising the amino acid sequence of SEQ ID No. 2, or a conservative variant polypeptide thereof, or an active fragment or derivative thereof.

2. The polypeptide according to claim 1, wherein the polypeptide is a polypeptide having the amino acid sequence of SEQ ID No. 2.

3. An isolated polynucleotide, characterized in that it comprises a nucleotide sequence selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide according to claim 1 or 2;

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

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

The polynucleotide according to claim 3, wherein the polynucleotide encodes a polypeptide having the amino acid sequence shown in SEQ ID No. 2.

5. The polynucleotide of claim 3, wherein the sequence of the polynucleotide is selected from the group consisting of:

(a) a sequence having positions 575 to 1849 in SEQ ID No. 1; and

(b) A sequence having positions 1 to 2507 in SEQ ID No. 1.

A recombinant vector comprising the polynucleotide according to claim 3, 4 or 5.

7. A genetically engineered host cell, characterized in that it is a host cell transformed or transduced with the vector of claim 6 or transformed or transduced with the polynucleotide of claim 3, 4 or 5. Host cells.

8. A method for preparing a polypeptide having human WW functional domain Huntington protein binding polypeptide activity, characterized in that the method comprises:

(a) Conditions suitable for expression of a WW domain-containing Huntington protein-binding polypeptide

-25-Replacement page (Article 26 糸) Culturing the host cell of claim 7; and

9. An antibody that specifically binds to the WW domain-containing Huntington protein-binding polypeptide of claim 1.

10. A method for screening a compound that mimics or regulates the activity or expression of a Huntington's protein-binding polypeptide containing a WW domain, characterized in that the polynucleotide of claim 1 or the polynucleotide of claim 3 is used.

11. A compound that mimics, promotes, antagonizes or inhibits the activity or expression of the polypeptide of claim 1 obtained by the method of claim 10.

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

13. The use of the compound according to claim 11, characterized in that the compound is used to modulate the activity of a WW domain-containing Huntington protein-binding polypeptide in vivo and in vitro.

14. A method for detecting a disease or susceptibility to a polypeptide-related disease according to claim 1, comprising:

(d) detecting abnormal expression of the polypeptide;

(e) detecting abnormal activity of the polypeptide; or

(f) detecting a change in a nucleic acid associated with abnormal expression or activity of the polypeptide.

15. A pharmaceutical composition comprising an effective amount of the polypeptide of claim 1 or the compound of claim 11 and a pharmaceutically acceptable carrier.

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