WO2001019864A1

WO2001019864A1 - Polynucleotides encoding novel human angiotensin ii-1 receptor proteins and the method of preparation and its use

Info

Publication number: WO2001019864A1
Application number: PCT/CN2000/000268
Authority: WO
Inventors: Yumin Mao; Yi Xie
Original assignee: Shanghai Biorigin Gene Development Co. Ltd.
Priority date: 1999-09-10
Filing date: 2000-09-11
Publication date: 2001-03-22
Also published as: CN1288057A

Abstract

The invention discloses a new kind of human angiotensin II-1 receptor proteins and polynucleotides encoding the polypeptides and a process for producing the polypeptides by recombinant methods. It also discloses the method of applying the polypeptides and polynucleotides for the prevention or treatment of various kinds of diseases, such as cardiovascular disease, coronary heart disease, essential hypertension and non-insulin dependent diabetes. The antibody and antagonist of the polypeptide and the therapeutic use of the same is also disclosed. In addition, it refers to the use of polynucleotides encoding novel human angiotensin II-1 receptor proteins.

Description

Polynucleotide encoding new human angiotensin I 1 -1 receptor related protein, and preparation method and application thereof

The present invention relates to a polynucleotide sequence encoding a novel human angiotensin II-1 receptor-related protein. The present invention also relates to a method for preparing the polynucleotide sequence and a protein encoded by the same, and uses of the polynucleotide and a protein encoded by the same. Background of the invention

Angiotensin II receptors (Ang II) is a vasoactive peptide that interacts with special membrane-bound receptors in target tissues (eg, kidneys, adrenals, blood vessels). At present, three types of angiotensin II receptors (types AT1, AT2, and AT3, respectively) such as type 1, type 2, and type 3 have been obtained. They all have seven hydrophobic transmembrane domain structures in structure and can be simultaneously Combined with a pair of Gq proteins, its main biological function is signal transduction. ATI and AT2 have 46% amino acid homology, of which ATI is composed of ATla and ATlb (both have the same coding region and different non-coding regions, the amino acid homology is higher than 90%), mainly in the heart of adults Vascular system, adrenal gland and intra-kidney expression are widely expressed in the entire body of the fetus and decline sharply with the birth of the fetus; AT2 is mainly expressed in the brain, adrenal medulla and ovary of adults. ATI's intracellular signal transduction is mainly achieved by activating lipase C to produce two results, namely the activation of protein kinase C and the fixation of Ca ²⁺ . Recently, it has also been found that tyrosine stimulation is also ATI signal transduction One way. Clinical studies have shown that ATI antagonists can cause an increase in the level of Angll in plasma and selectively stimulate AT2, and AT2 itself has an anti-ATI effect, so the function of AT2 as an ATI antagonist has important significance in clinical treatment [Miserey S, et al. Therapie 1998; 53: 205-11]. Angiotensin Π-1 receptor-related protein (hereinafter referred to as ATRAP) is a newly discovered ATI receptor-related protein. The molecular weight of ATRAP in mice is 18 kDa, and it only interacts with the carboxy-terminal cytoplasmic structure of ATla receptors. Domains, but not AT2, soothing kallikrein B2, β2 adrenergic receptors, etc. ATRAP mRNA is expressed in mouse kidney, heart, and testis, but not in mouse lung, liver, spleen, and brain. The interaction of ATRAP and ATla receptors can be determined by affinity chromatography, fluorescence microscopy, and specific immunoprecipitation of two proteins. Overexpression of ATRAP in COS-7 cells inhibits the activation of phospholipase C by the ATla receptor [Daviet L, et al., J Biol Chem 1999; 274: 17058-62].

As ATI is an important component of the renin-angiotensin system (RA system), it can mediate physiological effects such as vasomotor contraction, water and salt metabolism, and vascular smooth muscle proliferation and function regulation. It is a key step of the RA system acting on effectors. It is also one of the pathogenic genes of hypertension genetics. Therefore, ATRAP can detect and treat a variety of cardiovascular diseases by interacting with ATI, such as detecting and treating coronary heart disease and primary disease. Hypertension and non-insulin-dependent diabetes mellitus are particularly preferably used for the treatment of coronary heart disease. Therefore, the research and development of ATRAP polypeptides and their antibodies, agonists, and antagonists for therapeutic purposes are of great physiological and pharmacological significance (Soubrier F et al., Hypertension Journal, 1993,11. Suppl 5 : s20-27). Summary of invention

It is an object of the present invention to provide a novel isolated polypeptide of angiotensin II-1 receptor-related protein.

Another object of the present invention is to provide a polynucleotide sequence encoding an angiotensin type II-1 receptor-related protein.

Another object of the present invention is to provide a receptor comprising an angiotensin Π-1 type receptor. Recombinant expression vector of polynucleotide sequence of related protein.

It is another object of the present invention to provide a host cell comprising a recombinant expression body with a polynucleotide sequence encoding an angiotensin type II receptor-related protein.

Another object of the present invention is to provide a method for producing angiotensin type II-1 receptor-related protein.

Another object of the present invention is to provide antibodies and antagonists against the angiotensin type II-1 receptor-related protein of the present invention.

Another object of the present invention is to provide a method for diagnosing and treating a disease associated with an abnormal function of angiotensin type II-1 receptor-related protein.

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 embodiment of the present invention, the present invention provides a substantially pure angiotensin type II-1 receptor-related protein consisting essentially of the amino acid sequence shown in SEQ ID NO: 2. Angiotensin II-1 receptor-related proteins are characterized by a transmembrane domain. The polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a full-length polynucleotide sequence of 1108 bases, and its open reading frame is SEQ ID NO: 1 at positions 56-535, encoding a human angiotensin II-1 receptor-related protein of 159 amino acids. According to the amino acid sequence homology comparison, it was found that this polypeptide has 76% homology with ATRAP in rat testis tissue, and the polypeptide has conserved bases in the ATRAP gene family. It can be concluded that this new human ATRAP has the ATRAP gene family Similar 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 from other substances coexisting in the natural state. On, it is isolated and purified.

As used herein, "isolated angiotensin II-1 receptor-related protein or polypeptide" means that angiotensin II-1 receptor-related protein is substantially free of other proteins, lipids, sugars, or Other substances. Those skilled in the art can purify angiotensin II-1 receptor related protein using standard protein purification techniques. Substantially pure peptides can produce a single main band on a non-reduced polypropylene amidamine gel. The purity of angiotensin II-1 receptor related protein 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 is preferably a recombinant polypeptide.

The invention also includes fragments, derivatives and analogs of human angiotensin II-1 receptor related proteins. As used herein, the terms "fragment", "derivative" and

"Analog" refers to a polypeptide that retains substantially the same biological function or activity as the natural human angiotensin II-1 receptor-related protein of the invention. A polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide with another compound (such as a compound that extends the half-life of a polypeptide, such as (Polyethylene glycol), a polypeptide formed by fusion, or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (such as a leader sequence, a secretion sequence, or a sequence or protease sequence used to facilitate purification of the polypeptide). These fragments, derivatives and analogs are within the scope of those skilled in the art.

In another embodiment, the invention provides an isolated polynucleotide consisting essentially of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2. In another embodiment, the polynucleotide sequence of the present invention includes the nucleotide sequence shown in SEQ ID NO: 1. 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 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 thereof. A "degenerate variant" in the present invention refers to a polynucleotide that encodes a protein or polypeptide having SEQ ID NO: 2 but is different from the polynucleotide sequence shown in SEQ ID NO: 1 due to the degeneracy of the genetic code. sequence.

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

The term "polynucleotide encoding a polypeptide" refers to a polynucleotide that includes a polynucleotide encoding the polypeptide and a polynucleotide that includes 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 substantially 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 present invention also relates to a polynucleotide capable of hybridizing to a polynucleotide sequence described above under certain conditions, said sequence having at least 50%, preferably 70%, more preferably having 90% identity). The present invention particularly relates to a polynucleotide capable of hybridizing to a polynucleotide according to the present 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, 60; or (2) addition of denaturation during hybridization Agent, such as 50% (v / v) formamide, 0.1% calf serum /0.1% Ficoll, 42 "C, etc .; or (3) hybridization occurs only when the identity between the two sequences is at least 95% or more, and more preferably 97% or more. Furthermore, the hybridizable polynucleotide encodes The polypeptide 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 capable of hybridizing to the sequences described above. As used in the present invention, the "nucleic acid fragment" contains at least 15 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, most preferably at least 100 nucleotides. Nucleic acid fragments and above. Nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding angiotensin II-1 receptor-related proteins.

The present invention also relates to a vector comprising a polynucleotide of the present invention, a host cell produced by a genetic engineering method using the vector of the present invention, and a method for producing a polypeptide according to the present invention by recombinant technology.

Based on the disclosure of the present invention, the DNA sequence of the present invention can be obtained by various 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 with common structural characteristics .

Generation of specific DNA fragment sequences encoding angiotensin II-1 receptor-related proteins can also be obtained by the following methods: 1) Isolating double-stranded DNA sequences from genomic DNA; 2) Chemically synthesizing DNA sequences to obtain double-stranded polypeptides DNA.

Of the methods mentioned above, genomic DNA isolation is the most commonly used. When the entire amino acid sequence of the desired polypeptide product is known, a method of direct chemical synthesis of the DNA sequence can also be used. If the entire sequence of the desired amino acid is unclear, direct chemical synthesis of the DNA sequence is not possible. The alternative method is 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 Somatic cDNA library. There are many mature techniques for extracting mRNA, and kits are also commercially available (Qiagene). The 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 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 loss of function of a marker gene; (3) determination of the transcript of angiotensin II-1 receptor-related protein Level; (4) applying immunological techniques or measuring biological activity to detect the protein product of gene expression. The above methods can be used singly or in combination.

In the method (1), the probe used for hybridization should be homologous to any part of the polynucleotide of the present invention, and its length is at least 15 nucleotides, preferably 30 nucleotides, more preferably 50 Nucleotide, preferably more than 100 nucleotides. In addition, the length of the probe is usually within 2 kb, preferably within 1 kb. 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 genes or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioactive isotopes, fluorescein, or thallium (such as alkaline phosphatase). In the method (4), the protein product for detecting the expression of angiotensin II-1 receptor-related protein gene can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).

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 substance can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method. 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: 5463-5467). Such nucleotide sequence determination can also be performed using commercial sequencing kits and the like. To obtain the full-length cDNA sequence, sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice 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 recombinant angiotensin II-1 receptor-related protein (Science, 1984; 224: 1431). Generally there are the following steps:

(1) using the polynucleotide (or variant) encoding the human angiotensin II-1 receptor-related protein of the present invention, or transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide;

(2) a host cell cultured in a suitable medium;

(3) Isolating and purifying the protein of the present invention from a culture medium or a cell.

In the present invention, a polynucleotide sequence encoding an angiotensin type II-1 receptor-related protein may be inserted into a recombinant expression vector. The recombinant expression vectors used in the present invention include, but are not limited to, bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors. 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 and Lee) expressed in mammalian cells Nathans, Journal of Biochemistry 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 stably exist and replicate in the host. An important feature of expression vectors is that they usually contain an origin of replication, Movers, marker genes and translation control elements.

Methods known to those skilled in the art can be used to construct an expression vector containing an angiotensin Π-1 receptor-related protein-encoding DNA sequence and appropriate 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 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: the lac or trp promoter of E. coli; the lambda phage _PL promoter; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine-stimulated promoter, early and late SV40 promoter, retroviral LTRs and other known promoters that can 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, neomycin resistance, and green for eukaryotic cell culture. 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 so that they can 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 Sf; 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. Enhancer is DNA The cis-acting factor, usually about 10 to 300 base pairs, acts on a promoter 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 on the late side of the origin of replication, and adenovirus enhancers.

Those skilled 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 can be harvested after the exponential growth phase and treated with the CaCl ₂ method. The steps used are well known in the art. Alternatively, MgCl ₂ is used. If 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 and 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.

In the above method, the recombinant polypeptide of interest may be coated in a cell or 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. More specifically, these methods include, but are not limited to: conventional renaturation, salting-out methods, centrifugation, osmotic shock breaking bacteria, 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.

Recombinant angiotensin II-1 receptor-related proteins or peptides have many uses It is particularly preferably used for the detection and treatment of various cardiovascular diseases, such as the detection and treatment of coronary heart disease, essential hypertension, and non-insulin-dependent diabetes mellitus. These uses include, but are not limited to: direct use as a drug to treat diseases caused by angiotensin II-1 receptor-associated protein hypofunction or loss, and for screening to promote or fight angiotensin II-1 receptor-associated protein Functional antibodies, polypeptides or other ligands. For example, antibodies can be used to activate or inhibit the function of angiotensin II-1 type receptor-related proteins. Screening the peptide library with the expressed angiotensin II-1 receptor-related protein can be used to find therapeutic polypeptide molecules that can inhibit or stimulate the function of angiotensin II-1 receptor-related protein.

The invention also provides methods for screening compounds, especially for identifying compounds that increase (agonist) or suppress (antagonist) angiotensin II-1 receptor related proteins. These agonists or antagonists are particularly suitable for a variety of cardiac applications. Detection and treatment of vascular diseases, such as detection and treatment of coronary heart disease, essential hypertension and non-insulin-dependent diabetes. Agonists enhance biological functions such as intracellular signaling of angiotensin II-1 receptor-related proteins, while antagonists prevent and treat disorders related to intracellular signaling, such as various cancers. For example, mammalian cells or angiotensin type II-1 receptor-associated proteins can be cultured in the presence of drugs with labeled angiotensin II-1 receptor-related proteins. The ability of the drug to increase or suppress this interaction is then determined.

Antagonists of angiotensin Π-1 type receptor-related proteins include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human angiotensin II-1 receptor-related proteins can bind to and eliminate human angiotensin II-1 receptor-related proteins, or inhibit human angiotensin II-1 receptor-related proteins Production, or binding to the active site of the polypeptide prevents the polypeptide from performing its biological function. Antagonists of human angiotensin II-1 receptor-related proteins can be used to treat a variety of cardiovascular diseases, such as coronary heart disease, essential hypertension, and non-insulin-dependent diabetes mellitus. When screening compounds as antagonists, angiotensin II-1 receptor-related protein can be added to the bioanalytical assay, and the interaction between angiotensin II-1 receptor-related protein and its receptor can be influenced by the determination of the compound To determine if the compound is an antagonist. Receptor deletions and analogs that act as antagonists can be selected in the same way as the compounds selected above.

The polypeptide of the present invention can be used for peptide mapping analysis. For example, the polypeptide can be specifically cleaved physically, chemically, or obscuredly, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis.

A variety of methods are available for the production of antibodies against angiotensin II-1 receptor-related protein epitopes. 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.

Antibodies against angiotensin II-1 receptor-related proteins can be used in immunohistochemical techniques to detect angiotensin II-1 receptor-related proteins in biopsy specimens.

Monoclonal antibodies that bind to angiotensin II-1 receptor-related proteins 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 they metastasize.

The antibodies of the present invention can be used to treat or diagnose diseases related to angiotensin II-1 receptor-related proteins, and are particularly preferred for a variety of cardiovascular diseases, such as coronary heart disease, essential hypertension, and non-insulin-dependent diabetes mellitus. . Administration of an appropriate dose of antibody can stimulate or block the production or activity of angiotensin type II-1 receptor related proteins.

Antibodies can also be used to design immunotoxins that target a particular part of the body. For example, monoclonal antibodies with high affinity for angiotensin II-1 receptor-related proteins can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.). Together. A common method is to attack the amino group of an antibody with a sulfhydryl crosslinker such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill angiotensin II-1 receptors Related protein positive cells.

Polyclonal antibodies can be produced by immunizing animals such as rabbits, mice, and rats with angiotensin II-1 receptor-related proteins or peptides. A variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant.

Monoclonal antibodies to angiotensin II-1 receptor-related proteins can be produced by hybridoma technology (Kohler and Milstein. Ature, 1975, 256: 495-497). Chimeric antibodies that bind human constant regions to 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 angiotensin II receptor-related proteins.

Polypeptide molecules capable of binding to angiotensin II-1 receptor-related proteins can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. During screening, angiotensin II-1 receptor-related protein molecules must be labeled.

The polypeptide of the present invention can be used in combination with a suitable pharmaceutical carrier. 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. The person skilled in the art knows how to adapt these formulations to 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 instructional 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 manufacture, use, or sell them. In addition, the polypeptides of the invention can be used in combination with other therapeutic compounds. The pharmaceutical composition can be administered in a convenient manner, such as by topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. Angiotensin II-1 receptor-related protein is administered in an amount effective to treat and / or prevent a particular indication. The amount and dose range of angiotensin II-1 receptor-related protein to be administered to a patient will depend on many factors, such as the mode of administration, the natural conditions of the subject, and the judgment of the diagnostician.

The invention also relates to a diagnostic test method for quantitatively and locally detecting the level of angiotensin II-1 receptor-associated protein. These tests are well known in the art and include FISH assays and radioimmunoassays. Angiotensin II-1 receptor-associated protein levels detected in the test can be used to explain the importance of angiotensin II-1 receptor-associated proteins in various diseases and to diagnose diseases in which ATRAP can play a role.

Polynucleotides encoding angiotensin Π-1 type receptor-related protein can also be used for the diagnosis and treatment of angiotensin Π-1 type receptor-related protein-related diseases, and is particularly preferred for a variety of cardiovascular diseases such as detection and treatment Coronary heart disease, essential hypertension and non-insulin-dependent diabetes mellitus. For diagnosis, angiotensin II-1 receptor-related protein polynucleotides can be used to detect expression of angiotensin II-1 receptor-related protein or angiotensin II-1 receptor-related protein in disease states Abnormal expression. For example, the DNA sequence of angiotensin II-1 receptor-related protein can be used to hybridize biopsy specimens to determine the abnormal expression of angiotensin II-1 receptor-related protein. Hybridization techniques include Southern blotting, Northern blotting, in situ hybridization, and the like. 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 or a DNA chip for analyzing differential expression analysis and gene diagnosis of genes in a tissue. Angiotensin II-1 receptor-related protein-specific primers can be used for RNA-polymerase chain reaction (RT-PCR) in vitro amplification to detect the transcription products of angiotensin II-1 receptor-related protein. Detecting mutations in the angiotensin II-1 receptor-related protein gene can also be used to diagnose angiotensin II-1 receptor-related protein-related diseases, and is particularly preferred for a variety of cardiovascular diseases such as detecting and treating coronary heart disease, Essential hypertension and non-insulin-dependent diabetes mellitus. Angiotensin II-1 receptor-associated protein mutations include point mutations, translocations, deletions, recombination, and any other abnormalities compared to the DNA sequence of normal wild-type angiotensin II-1 receptor-related proteins. 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 the 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) can be used to label the chromosomal position of the beam. According to the present invention, the action of D N A on chromosomes is an important first step in correlating these sequences with disease-related solids.

In short, by preparing PCR primers (preferably 15-25 bp) from c D N A, the sequence can be mapped to chromosomes. The computer analysis of c D N A quickly selected primers that did not cross an exon of the genome D N A, thereby complicating the amplification procedure. These primers were then used for PCR to screen somatic hybrid cells containing a single human chromosome. Only these hybrid cells containing human genes corresponding to the primers will produce amplified fragments.

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

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

Once the sequence is accurately mapped to a specific location on the chromosome, the physical location of the sequence on the chromosome can be correlated with the gene map data. These data can be found in, for example, V. Mckusick, Mendelian Inheritance in Man I (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship of genes to diseases that have been mapped to some chromosomal regions.

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 precise location of c DNA in chromosomal regions associated with disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping Resolution and every 20kb—genes).

Comparing diseased 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 with a PCR based on the c D N A sequence. Finally, the complete sequencing of the genes of several individuals is needed to confirm the existence of mutations and the differences between mutations and polymorphisms.

Angiotensin II-1 receptor-related protein polynucleotides can also be used for a variety of therapeutic purposes, and are particularly preferred for a variety of cardiovascular diseases, such as detection and treatment of coronary heart disease, essential hypertension, and non-insulin-dependent diabetes. . Gene therapy technology can be used to treat cell proliferation caused by angiotensin II-1 receptor-related protein expression or abnormal / inactive angiotensin II-1 receptor-related protein expression Colonization, development or metabolic abnormalities. Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant angiotensin II-1 receptor-related proteins and to inhibit endogenous angiotensin II-1 receptor-related protein activity. For example, a variant angiotensin II-1 receptor-associated protein may be a truncated angiotensin II-1 receptor-associated protein that lacks a signaling domain, although it can bind to downstream substrates But lacks signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of angiotensin II-1 receptor-related protein. Virus-derived expression vectors such as retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, and parvovirus can be used to transfer angiotensin II-type 1 receptor-related protein genes into cells. Constructed to carry angiotensin

Methods for recombinant viral vectors of type II-1 receptor-associated protein genes can be found in existing literature (Sambrook, et al.). In addition, the recombinant angiotensin II-1 receptor-related protein gene can be packaged into liposomes and transferred into cells.

Oligonucleotides (including antisense R A and DNA) and ribozymes that inhibit angiotensin II-1 receptor-related protein mRNA are also within the scope of the present invention. A ribozyme is an enzyme-like RNA molecule that specifically cleaves specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation. Antisense RNA, DNA, and ribozymes can be obtained by any existing technology for synthesizing RNA or DNA. For example, solid-phase phosphate amide chemical synthesis technology has been widely used for oligonucleotides. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA sequence has been integrated downstream of the vector's RNA polymerase promoter. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphothioester or peptide bonds instead of phosphodiester bonds.

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 in vitro through a vector (such as a virus, phage, or plasmid), and then introducing the cell Transplanted into the body, etc. The polypeptides and polynucleotides of the present invention also have other uses. For example, the polypeptides of the present invention can be used for identification of peptide fingerprints, and the coding sequences or fragments thereof of the present invention can be used to make gene chips or microarrays. These applications will be apparent to those skilled in the art from the teachings of the present invention. The following examples will further illustrate the present invention, but not to limit the present invention. Examples

Example 1: Cloning of a polynucleotide encoding a human angiotensin II-1 receptor-related protein

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 (Qiagene). 2 g poly (A) mRNA was reverse transcribed to form cDNA. Smart cDNA Cloning Kit (purchased from Clontech) was used to orient the cDNA fragment into At the multiple cloning site of the vector, E. coli DH5a was transformed to form a cDNA library. A total of 3028 clones were obtained. The dideoxy method was used to determine the sequence of 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 (0894e01) was new DNA. The DNA sequence contained in the 0894e01 clone was determined in both directions by synthesizing a series of primers. Computer analysis shows that the full-length cDNA contained in the 0894e01 clone is a new DNA sequence (as shown in Seq ID Nol), and has a 479bp ORF from 56th to 535th positions, encoding a new protein (such as Seq ID No 2), the sequence is named pBS-0894e01. This protein is named human angiotensin II-1 receptor-related protein. Example 2: Cloning of human angiotensin Π-1 receptor-related egg by RT-PCR White:

CDNA was synthesized using fetal brain cell total RNA as a template and oligo-dT as a primer for reverse transcription reaction. After purification with Qiagen's kit, PCR amplification was performed with the following primers: Forward primer Fl 5, -GGGAGCCTAGGAGCCCCC-3, located at Start of SEQ ID NO1; reverse primer R1: 5'- GAATTCCCCAAACTTTAATG-3 'is located at 1088-1108bp of SEQ ID NO1. Amplification reaction conditions: 50 mmol / L KCl, 10 mmol / L Tris-Cl, (pH 8.5), 1.5 mmol / L MgCl ₂ , 20 (^ mol / L dNTP, 25 pmol primers, 2.5 in a reaction volume of 50 μ1) Taq DNA polymerase of U. Reaction on the PE9600 DNA thermal cycler under the following conditions for 25 cycles: 94TC for 30 seconds; 55X for 30 seconds; 72 X for 2 minutes. Set β-actin at the same time during RT-PCR The positive control and template blank were negative controls. After purification of the amplified product QIAGEN kit, the TA cloning kit was used to connect to the PCR vector (Invitrogen), and the DNA sequence was determined. Results The DNA sequence of the PCR product was the same as that of SEQ ID NO1. -1108bp is exactly the same. Example 3, Homologous search of cDNA clones

The polynucleotide sequence of the human angiotensin II-1 receptor-related protein provided by the present invention and its encoded protein sequence are compared with databases of Genbank ^ Swissport and other databases for homology. The program used for searching is Blast (Basic local Alignment search tool) (1993, Proceedings of the National Academy of Sciences, 90: 5873-5877), Blast can find many homologous genes, among which the gene with the highest homology with the gene of the present invention, the protein encoded by it is in the Genbank standard. Accession number is AF102548. These retrieved gene or protein sequences can be retrieved from the Genbank database. The recalled sequences can be compared using the Pileup (multi-sequence) and Gap (two-sequence) programs in the GCG software package. Protein function predictions can be analyzed using the Motif program. The results of the homology search show that the human angiotensin II-1 receptor-related protein provided by the present invention is related to the rat angiotensin II-1 receptor The protein has 76% homology, and the comparison of isogenicity is as follows.

> gb | AAD25997.1 | AF102548_l (AF102548) ATI receptor-related protein [Mus musculus]

Length = 161 Score = 622 (219.0 bits), Expectation = 9.7e-61, P = 9.7e-61

Identity = 123/160 (76%), Similarity = 134/160 (83%)

Query: 1 MELPAVNLKVILLGHWLLTTWGCIVFSGSYAWANFTILALGVWAVAQRDSIDAISMFLGG 60

MELPAVNLKVILL HWLLTTWGC + VFS SYAW NFTILALGVWAVAQRDSIDAI MFLGG Sbjct: 1 MELPAVNLKVILLVHWLLTTWGCLVFSSSYAWGNFTILALGVWAVAQRDSIDAIGMFLGG 60

Query: 61 LLATIFLDIVHISIFYPRVSLTDTGRFGVGMAILSLLLKPLSCCFVYHMYRERGGELLVH 120

L + ATIFLDI ++ ISIFY V + DTGRFG GMAILSLLLKP SCC VYHM + RERGGEL + Sbjct: 61 LVATIFLDIIYISIFYSSVATGDTGRFGAGMAILSLLLKPFSCCLVYHMHRERGGELPLR 120

Query: 121 TGFLGSSQDRSAYQTIDSA-EAPADPFAVPEGRSQDA-RG 158

F G SQ + SAYQTIDS + + A ADPFA E + Q A RG

Sbjct: 121 PDFFGPSQEHSAYQTIDSSSDAAADPFASLENKGQAAPRG 160 Example 4: Northern blot analysis of human angiotensin type II-l receptor-related protein gene expression

Total RNA was extracted in one step [Anal. Biochem 1987, 162, 156-159]. This method involves acid guanidinium thiocyanate phenol-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) were added. ), Mix and centrifuge. Aspirate the aqueous layer, add isopropanol (0.8 vol) and mix Centrifuge to obtain RNA pellet.

The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water. Electrophoresis was performed on a 1.2% agarose gel containing 2 g of RNA on 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-ImM EDTA-2.2M formaldehyde. Then transferred to nitrocellulose. the probe (about 2xl0 ⁶ cpm / ml) ³² P- labeled at 42 in a solution "hybridization overnight, the solution containing 50% formamide -25mM KH ₂ P0 ₄ (pH7. 4) -5xSSC-5xDenhardt, s solution and 200 g / ml salmon sperm DNA. Α- ³² P dATP was used to prepare ³² P-labeled DNA probes by random primer method. The DNA probes used were human blood vessels amplified by PCR. The sequence of angiotensin II-1 receptor-related protein coding region or a fragment thereof. After hybridization, the filter was washed in lxSSC-0.1% SDS at 65 for 30 minutes. Then, it was analyzed and quantified by Phosphor Imager '

The results showed that the human angiotensin II-1 receptor-related protein gene can be specifically expressed in testis tissue. Example 5: In vitro expression, isolation and purification of recombinant human angiotensin II-1 receptor-related protein

A pair of primers were designed at the start codon and the stop codon of the angiotensin type II-1 receptor-related protein gene, respectively, with 5 and ends carrying BamHI and EcoRI restriction sites. The plasmid pBS-0894e01 was used as a template for PCR amplification to obtain the coding region of angiotensin II-1 receptor-related protein gene. After the digestion, the amplified fragment was inserted into the expression vector PGEX-2T (purchased from Pharmacia Biotech) and transformed into E. coli DH5a. On the LB plate containing ampicillin and IPTG, five white recombinant transformants were screened for DNA sequence analysis. The result is exactly the same as the gene coding region sequence in pBS-0894e01. The recombinant clone can express the GST-ATRAP fusion protein, and the clone number is pATRAP.

Pick a loop of E. coli DH5a strain and inoculate it in 20ml LB medium (containing ampicillin 100ug / ml), 37 "C shake culture overnight as the seed liquid, take the seed solution and transfer it to 4 liters of LB medium at 2% inoculation amount, 37" C shake culture until the cell A ₆₀₀ = 0.7 (logarithmic (Growth period), add IPTG to a final concentration of 0.4mmol / L, and incubate for 25 * culture for 12 hours, collect the cells by centrifugation, wash with lxPBS with buffer A (16mM Na ₂ HP0 ₄ , 4mM NaH ₂ P0 ₄ , pH 6.5 ) Dissolve at 10ml / g, sonicate in a water bath, collect the supernatant after centrifugation, pass the supernatant through a glutathione-Sepharose 4B chromatography column, rinse with buffer A, and then use 5mM reduced The eluate of glutathione was eluted. The eluate showed about 17.5 kDa (with GST-ATRAP fusion protein product) by SDS-PAGE. Example 6: Preparation of antibodies against angiotensin Π-1 receptor-related protein

Polypeptide synthesizer (PE-ABI) was used to synthesize the following angiotensin II-1 receptor-related protein-specific polypeptide: Ala-Pro-Ala-Asp-Pro-Phe-Ala-Val-Pro-Glu (p. 141) -150 digits). The polypeptide 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 plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once. A titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum. Protein A-Sepharose was used to isolate total IgG from antibody-positive rabbit serum. The peptide was bound to a cyanogen bromide-activated Sepharose 4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography. The immunoprecipitation method demonstrated that the purified antibody can specifically bind to the angiotensin type II-1 receptor-related protein of the present invention. Example 7: Yeast two-hybrid experiment

Plasmid preparation:

Mouse ATla receptor (: terminal cytoplasmic domain (ATla C-ter; amino acid 297-359) The Gal4 binding domain fused to the yeast shuttle vector pBD-Gal4 (Stratagene) is amplified by PCR. In the same way, ATla receptor tails (ATla C-ter 349, 339, and 329) were subcloned into pBD-Gal4 by PCR amplification using antisense primers containing specific site stop codons.

Using the ^ 0 tail of 11 ^, using an XhoI- (dT) 18 primer and an EcoR I linker, angiotensin type II-1 receptor-related protein was inserted into the plasmid pAD-Gal4 (Stratagene). Two-hybrid screening:

Yeast strain YRG-2 (Stratagene) containing two Gal4-induced reporter genes (HIS3 and LacZ) and ATla C-ter angiotensin Π-1 type receptor-related protein hybrid expression plasmid were co-transcribed. Defective (Try-Leu'His) medium was cultured for three days, His + colonies were added to the selective medium, and nitrocellulose membrane culture was performed to measure the P-galactosidase activity. The results are shown in Table 1, Table 1 and ATRAP and ATla receptors. Tail deletion mutant interactions

Gal4 binding domain hybrids Gal4 active domain hybrids Gal unit ° /. Combined%

ATla C-ter wild type ATRAP 160 100

ATla C-ter △ 349 ATRAP 60 37.5

ATla C-ter △ 339 ATRAP 0 0

ATla C-ter Δ 329 ATRAP 0 0

Claims

1. An isolated human angiotensin II-1 type receptor-related protein comprising a polypeptide having the amino acid sequence shown in SEQ ID No. 2, or a conservative variant thereof, or an active fragment thereof, or an activity derivative thereof Thing.

The polypeptide according to claim 1, which is a polypeptide having an amino acid sequence shown in SEQ ID No. 2.

3. An isolated polynucleotide comprising a nucleotide sequence that is up to 70% identical to 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 the polynucleotide in (a).

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

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

(a) a polynucleotide sequence having positions 53 to 535 in SEQ ID No. 1;

(b) a polynucleotide sequence having positions 1 to 1108 in SEQ ID No. 1.

A vector comprising the polynucleotide according to claim 3.

7. A genetically engineered host cell, which is a host cell selected from the group consisting of:

(a) a host cell transformed or transduced with the vector of claim 6;

(b) a host cell transformed or transduced with the polynucleotide of claim 3.

8. A method for preparing a polypeptide having human angiotensin II-1 type receptor-related protein activity, the method comprising:

(a) culturing the host cell according to claim 7 under conditions suitable for expression of angiotensin II-1 type receptor-related protein;

(b) Isolation of angiotensin II-1 type receptor-associated protein activity from culture Of peptides.

An antibody capable of specifically binding to a human angiotensin II-1 receptor-related protein according to claim 1.

10. A method for screening a compound that mimics, promotes, antagonizes or inhibits the activity of the human angiotensin II-1 type receptor-related protein according to claim 1, comprising using the polypeptide according to claim 1.

11. A compound obtained according to the method of claim 10, which has an activity that mimics, promotes, antagonizes or inhibits the human angiotensin II-1 type receptor-related protein according to claim 1.

12. A method of using the compound of claim 11 to modulate the activity of angiotensin II-1 type receptor-related protein protein in vivo and in vitro.

13. A method for detecting a disease or susceptibility to a disease associated with abnormal expression of angiotensin Π-1 type receptor-associated protein according to claim 1, characterized in that it detects an angiotensin II encoding the angiotensin II according to claim 1 -Mutations in the nucleic acid sequence of type 1 receptor-related proteins.

14. The use of the polypeptide according to claim 1, for screening an agonist that promotes angiotensin II-1 type receptor-related protein activity, or for screening for angiotensin II-1 type receptor-related protein activity Antagonists are also used for peptide fingerprinting.

15. A pharmaceutical composition comprising a therapeutically effective amount of the polypeptide of claim 1 and a pharmaceutically acceptable carrier.

16. The use of a polypeptide according to claim 1 or a compound according to claim 11 for the preparation of a pharmaceutical composition for treating a disease associated with an angiotensin II-1 receptor-associated protein activity abnormality.

17. The use according to claim 16, wherein the diseases are coronary heart disease, essential hypertension and non-insulin-dependent diabetes.