WO2001019999A1 - A GENE ENCODING A NOVEL THREONYL-tRNA SYNTHETASE, ITS USES AND THE PREPARING METHODS - Google Patents

Abstract

The present invention relatives to a polynucleotide sequence encoding a novel human threonyl-tRNA synthetase, the invention also relatives to the methods for preparing said polynucleotide and the protein it encoded by recombinant technology, the invention discloses the uses of the human threonyl-tRNA synthetase and the polynucleotide sequence encoding said polypeptide in preparing pharmaceutical composition for treating to the diseases resulting from low or lack of function of human threonyl-tRNA synthetase, such as various tumors. The invention also discloses the methods for preparing antibodies against said polypeptides, screening agonists, as well as their uses in treatment.

Description

 Gene encoding a new threonine transfer ribonucleic acid synthetase

 FIELD OF THE INVENTION

 The present invention relates to a polynucleotide sequence encoding a novel human threonyl transfer ribonucleic acid synthetase. The present invention also relates to a method for preparing the polynucleotide and a protein encoded by the same, and the polynucleotide and the encoding Use of protein. Background of the invention

Aminoacyltransferases are a large family of enzymes. In prokaryotes there are at least twenty different forms of amino acid aminoamidine transfer ribonucleic acid synthetases, while in eukaryotes there are only two forms. Amino acid aminoacyltransferase synthetase, namely: cytosolic and mitochondrial type, they have a common quaternary structure, and their subunits have multiple changes [Schimmd P. Annu. Rev. Biochem.) 56: 125-158 (1987)]. Aminoacyltransferase synthetases can be divided into two groups: Group I consists mainly of kinases and dehydrogenases, and the remaining synthetases are grouped into Group II. The II enzyme has three characteristic sequence motifs, motif 1 is the dimer interface, and motifs 2 and 3 are active sites. When the amino acid and ATP binding sites undergo a contact reaction, the II enzyme is combined into an enzyme. This results in a structural fold, which is different from the Rossmann fold pattern of group I. The second group of alcohols can be further subdivided into groups 2a (proline, threonine, histidine, serine, etc.) and group 2b (aspartic acid, aspartyl, and lysine, etc.) ), In addition to the common characteristics of Group II synthetic alcohols mentioned above, each has some important motifs [Cusack S. et al. Nucleic Acids Res 1991 19 (13): 3489-98]. Both sets of aminoacyltransferase synthetases can activate amino acids. In the first step of protein biosynthesis, the activated amino acids are transferred to specific tRNA molecules, but they are attached to ATP. Different sites: Group I enzymes are attached to 2, hydroxyl groups, and group II enzymes are attached to 3, hydroxyl groups [Eriani G. et al., J Mol Evol. 1995 40 (5): 499- 508] .

 Threonyltransferase synthetase (hereinafter abbreviated as thrS) is one of the members of the large family of aminotransferase synthetases, and it has the structure and function of the aforementioned aminoacyltransferase synthesis. Human threonyl transferase synthase and threonine transfer ribonucleic acid synthase found in cytoplasm, yeast mitochondria, and B. subtilis have high homology. A detailed understanding of threonyl transfer ribonucleic acid synthase and its signal transduction pathways of S. aureus has revealed some mechanisms of inflammatory diseases and uncontrolled proliferative diseases [Hodgson, et al., U.S. Patent, 5795757]. The human threonyl transfer ribonucleic acid synthetase cDNA, oligonucleotides, peptides, and antibodies thereof provided by the present invention are useful for studying signal transduction in different tissues and cells, and for diagnosing diseases related to threonine transfer ribonucleic acid synthase disorders It is of great value to classify threonine transfer ribonuclease synthetase inhibitors or drugs used to treat these diseases. Summary of invention

 It is an object of the present invention to provide an isolated threonyl transfer RNA for synthesis.

 Another object of the present invention is to provide a polynucleotide sequence encoding a threonine transfer ribonucleic acid synthetase.

 Another object of the present invention is to provide a recombinant expression vector containing a polynucleotide encoding a threonine transfer ribonucleic acid synthetase.

 Another object of the present invention is to provide a host cell comprising a recombinant expression vector having a polynucleotide encoding a threonyl transfer ribonucleic acid-synthesizing alcohol.

Another object of the present invention is to provide a method for producing threonine transfer ribonucleic acid synthetase. Another object of the present invention is to provide an antibody against the threonine transfer ribonucleic acid synthetase of the present invention.

 Another object of the invention is to provide antagonists against the polypeptides of the invention. Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormal function of threonyl transferase synthetase.

 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 a first embodiment, the present invention provides a substantially pure threonyl transfer ribonucleic acid synthetase consisting essentially of the amino acid sequence shown in SEQ ID NO: 2. Threonyl transfer ribonucleic acid synthesis is characterized by having a glutamyl transfer ribonucleic acid synthetase domain.

 As used in the present invention, "substantially pure" is threonine transfer ribonucleic acid synthesizer which is essentially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. Those skilled in the art can purify threonine transfer ribonucleic acid synthetase using standard protein purification techniques. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of threonine transfer RNA synthetase 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 according to the present invention.

As used in the present invention, the polypeptide of the present invention such as a fragment, derivative or analog of SEQ ID NO: 2 may be: (I) a polypeptide in which one or more amino acid residues Group is substituted by a conservative or non-conservative amino acid residue (preferably a conservative amino acid residue), and the substituted amino acid may or may not be encoded by a genetic codon; or (II) a polypeptide in which one or more amino acids The residue contains a substituent; or

(III) a polypeptide in which the mature polypeptide is fused to another compound; or

(IV) A polypeptide in which an additional amino acid is fused to a mature polypeptide, which is 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 invention provides an isolated 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 2741 bases in length and its open reading frame encodes 718 amino acids. According to the amino acid sequence homology comparison, it was found that this polypeptide has 61% homology with thrS in yeast mitochondria, and the polypeptide has conserved bases in the thrS gene family, which can be inferred from the synthesis of this new human threonyl transfer ribonucleic acid The enzyme has a similar structure and function of the thrS gene family.

 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 a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant. As used herein, a "degenerate variant" is It refers to a nucleic acid sequence that encodes a protein or peptide having SEQ ID NO: 2, but is different from the coding region sequence shown in SEQ ID NO: 1 due to the coexistence of codons.

 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); and Non-coding sequence.

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

 The invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention. This variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. 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, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide it encodes.

 The invention also relates to a polynucleotide capable of hybridizing to the sequence described above (there is at least 70%, preferably 80%, more preferably 90%, or 95% identity between the two sequences). The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "strict conditions" means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 "; or (2) added during hybridization Denaturing agents, such as 50% (v / v) formamidine, 0.1% calf serum / 0.1% Ficoll, 42, etc .; or (3) only the identity between the two sequences is at least 95% or better, preferably Hybridization occurs only when it is above 97%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.

The invention also relates to nucleic acid fragments that hybridize to the sequences described above. As used in the present invention, a "nucleic acid fragment" is at least 15 nucleotides in length, preferably at least 15 nucleotides in length. 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nucleotides. Nucleic acid fragments can be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding threonyl transfer ribonucleic acid synthetase.

 The present invention also relates to a vector comprising a polynucleotide sequence 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.

 The DNA sequence of the present invention can be obtained by several methods. For example, DNA is isolated using hybridization techniques 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 common structural features .

The production of specific DNA fragment sequences encoding threonyltransferases 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-as described above Of the methods mentioned, genomic DNA isolation is the most commonly used. When the entire amino acid sequence of the desired polypeptide product is known, direct chemical synthesis of the DNA sequence is also an alternative method. If the entire sequence of the required number of amino acids is unclear, direct chemical synthesis of the DNA sequence is not possible, and the method chosen is the isolation of the cDNA sequence. The standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a scutellum or phage 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 combined with polymerase chain reaction technology, even very small 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-RNA hybridization; (2) the presence or loss of function of a marker gene; (3) determination of the level of a threonyl transfer ribonucleic acid-synthesized transcript (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 is a nucleotide sequence that is homologous to any part of the polynucleotide of the present invention, and has a length of at least 15 nucleotides, preferably 20-30 nuclei. The nucleotide is preferably 50 to 60 nucleotides, and more preferably 100 nucleotides or more. The probe used here is usually a DNA sequence chemically synthesized based on the DNA sequence information of the gene of the present invention. The gene itself or a fragment of the present invention can of course be used as a probe. DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).

 In the (4) method, immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product expressed by the threonyltransferase synthetase gene expression.

 The method of using the 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 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 or various DNA fragments and the like obtained as described above can be measured by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). This type of nucleotide sequence determination is also available Industry sequencing kits. In order 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 threonine transfer ribonucleic acid synthetase (Science, 1984; 224: 1431). Generally there are the following steps:

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

 (2) host cells cultured in a suitable medium;

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

 In the present invention, the threonine transfer ribonucleic acid synthetase 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; pMSX D expression vectors (Lee) expressed in mammalian cells And 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 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 an expression vector containing a threonyltransferase synthetase-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 laboraty. New York, 1989). Said The DNA sequence is operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters are: E. coli lac or trp promoter; lambda phage p _L promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine promoter, early and late SV40 Promoters, retroviral LTR promoters, 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 the selection of transformed host cells, such as dihydrofolate reductol for use in 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 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 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. 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 adenoviral enhancers.

Those of ordinary skill in the art know how to choose an appropriate vector, promoter, 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, the preparation of competent cells capable of absorbing DNA is generally harvested from 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 gallate co-precipitation method, conventional mechanical methods such as obvious sign injection, 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.

 The recombinant polypeptide required in the above method is coated intracellularly, extracellularly, or expressed on the cell membrane or secreted extracellularly. If necessary, the physical, chemical, and other properties can be used to isolate and purify the recombinant protein by various separation methods. These methods are well known to those skilled in the art. More specifically, conventional renaturation, treatment with a protein precipitant (salting out method), centrifugation, osmotic lysis, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), and adsorption chromatography can be mentioned. , Ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

Recombinant threonyl transfer ribonucleic acid synthetase protein or polypeptide has many uses. These uses include (but are not limited to) direct use as a drug to treat diseases caused by low or loss of threonyltransferase synthase function, and to screen out antibodies that promote or counteract threonyltransferase synthase function, Polypeptide or other ligand. For example, antibodies can be used to activate or inhibit the function of a threonyltransferase synthetase. / NOO / 0 75 Selecting a peptide library using the expressed recombinant threonyltransferase synthetase protein can be used to find therapeutic polypeptide molecules that can inhibit or stimulate threonyltransferase synthetase function.

 The invention also provides methods for screening drugs to identify agents that increase (agonist) or inhibit (antagonist) threonine transfer ribonucleic acid synthetase. Agonists enhance biological functions such as threonyltransferase-stimulating cell proliferation, while antagonists block and treat disorders related to excessive cell proliferation, such as various cancers. For example, mammalian cells or membrane preparations expressing threonyltransferase synthetase can be cultured with labeled threonyltransferase synthetase in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.

 Antagonists of threonyltransferase synthetase proteins include selected antibodies, compounds, receptor deletions, and the like. Antagonists of human threonyltransferase synthetase protein can bind to human threonyltransferase synthase protein and eliminate its function, or inhibit the production of human threonyltransferase synthetase protein, or The active site binding of the polypeptide prevents the polypeptide from performing its biological function.

 When screening compounds as antagonists, this new human threonine transfer ribonucleic acid synthesis protein can be added to a bioanalytical assay to determine whether the compound affects this new human threonyl transferase RNA synthesis alcohol protein and Interactions between its receptors determine whether a compound is an antagonist. In the same way as the above-mentioned compounds are selected, receptor deletions and analogues that act as antagonists can be screened.

 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 enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis.

A variety of methods can be used to produce antibodies against threonine transfer ribonucleic acid synthase 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 threonyltransferase synthetase can be used in immunohistochemical techniques to detect threonyltransferase synthetase in biopsy specimens.

 Monoclonal antibodies that bind to threonyltransferase synthetase 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 diseases related to threonine transfer ribonucleic acid synthetase. Administration of an appropriate dose of the antibody can stimulate or block the production or activity of threonyl transfer ribonucleic acid synthetase.

 Antibodies can also be used to design immunotoxins that target a particular part of the body. For example, threonyltransferase synthetase-rich monoclonal antibodies can covalently bind to bacterial or phytotoxins (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 threonyl transfer ribonucleic acid synthase Cell.

 Polyclonal antibodies can be produced by immunizing animals such as rabbits, mice, rats, etc. with threonyltransferase synthetase protein or peptides. A variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant.

 Monoclonal antibodies against threonyltransferase synthetase can be produced by hybridoma technology (Kohler and Milstein. Nature, 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. Patent No. 4,946,778) can also be used to produce single-chain antibodies against threonyltransferase synthetase.

Polypeptide molecules capable of binding to threonine transfer ribonucleic acid synthesis alcohol can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase Got. During enrollment, threonine transfer ribonucleic acid synthetase 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. 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 regulatory agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government regulatory agencies that produce, use, or sell. In addition, the polypeptides of the invention can be used in combination with other therapeutic compounds.

 The pharmaceutical composition can be administered in a convenient manner, such as by topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. Threonyltransferase synthetase is administered in an amount effective to treat and / or prevent a specific indication. The amount and range of threonyltransferase synthesis to be administered to a patient 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 qualitative detection of threonine transfer ribonucleic acid synthase levels. These tests are well known in the art and include FLISH assays and radioimmunoassays. The level of threonyltransferase synthetase detected in the test can be used to explain the importance of threonine transfer ribonucleic acid synthetase in various diseases, and to diagnose the role of threonyltransferase synthetase. disease.

Polynucleotides encoding threonine transfer ribonucleic acid syntheses can also be used in the diagnosis and treatment of threonyl transfer ribonucleic acid synthase-related diseases. For diagnostic purposes, a polynucleotide encoding a threonyltransferase synthetase can be used to detect the expression of threonyltransferase synthetase or to synthesize threonyltransferase under disease conditions. Abnormal expression of enzymes. For example, a polynucleotide sequence encoding a threonine transfer ribonucleic acid synthetase can be used to hybridize biopsy specimens to determine abnormal expression of threonyl transfer ribonucleic acid synthesis. 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 (Microarray) or a DNA chip (DNA Chip) for analyzing differential expression analysis and gene diagnosis of genes in a tissue. Tranylase synthetase-specific primers for RNA-polymerase chain reaction (RT-PCR) amplification in vitro can also detect the transcription product of threonyltransferase synthetase.

 Detection of mutations in the gene encoding threonyltransferase synthetase can also be used to diagnose diseases related to threonyltransferase synthesis. Threonine transfer ribonucleic acid synthetase mutations include point mutations, translocations, deletions, recombination and any other abnormalities compared to the normal wild-type threonyltransferase DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect 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 on a human chromosome and can hybridize to it. Specific sites on chromosomes need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome positions. According to the present invention, an important first step in correlating these sequences with disease-related genes is to locate these DNA sequences on the chromosome.

In short, PCR primers (preferably 15-25 bp) are prepared from c DNA, and the sequences can be mapped on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells that contain the human gene corresponding to the primer will produce amplified fragments. PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes. Using the oligonucleotide primers of the present invention, by a similar method, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization. Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct a chromosome-specific c DNA library.

 Fluorescent in situ hybridization (FISH) of c D N A 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 located at the exact chromosomal location, the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendelian Inheritance in Man (available online with Johns Hopkins University Welch Medical Librar). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.

 Next, the difference in c D N A or genomic sequence between affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individual, the mutation may be the cause of the disease. Comparing diseased and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable with a PCR based on the c D N A sequence.

The resolution capabilities of current physical mapping and gene mapping technology are accurately mapped to the c DNA of the chromosomal region associated with the disease, which can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene). Polynucleotides encoding threonyltransferase synthetases can also be used for a variety of therapeutic purposes. Gene therapy technology can be used to treat abnormal cell proliferation, development, or metabolism due to non-expression or abnormal / inactive threonyltransferase synthetase expression. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated threonyltransferase synthetase to inhibit endogenous threonyltransferase synthetase activity. For example, a variant threonine transfer ribonucleic acid synthetase may be a truncated threonine transfer ribonucleic acid synthase that lacks a signaling domain, although it can bind to downstream substrates, but lacks signal transduction activity. . Therefore, the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of threonine transfer ribonucleic acid. Virus-derived expression vectors such as retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer the threonyl transfer ribonucleic acid synthetase gene into cells. Methods for constructing recombinant viral vectors carrying threonine transfer ribonucleic acid synthetase genes can be found in existing literature (Sambrook et al.). In addition, the recombinant threonyltransferase synthetase gene can be packaged into liposomes and transferred into cells.

Oligonucleotides (including antisense RNA and DNA) and ribosomes that inhibit threonine transfer ribonucleic acid synthetase mRNA are also within the scope of the present invention. Ribo alcohol is an enzyme-like RNA molecule that can specifically decompose specific RA. Its mechanism of action is that the nucleophilic 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, 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 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 phosphorothioate or peptide bond is used for the linkage between the ribonucleosides instead of the 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, and then transplanting the cell Into the body and so on.

 The following examples will further illustrate the invention, but are not intended to limit the invention. Examples

Example 1: Cloning of cDNA encoding threonyltransferase synthetase:

 Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / pan / chloroform. Poly (A) mRNA was isolated from total RNA using Quik mRNA Isolation Kit (Qiegene). 2ug poly (A) mRNA was formed into cDNA by reverse transcription. The Smart cDNA cloning kit (purchased from Clontech) was used to insert the cDNA fragment into the multi-cloning site of the vector and transformed into E. coli DH5a to form a cDNA library. A total of 3028 clones were obtained. The 5 'and 3' ends of all clones were determined by dideoxy method. The determined cDNA sequence was compared with the existing public DNA sequence database, and it was found that the DNA sequence of one clone (215H11) was new. A series of primers were synthesized to determine the DNA sequence of the 215H11 clone in both directions. Computer analysis showed that the full-length cDNA contained in the 215H11 clone was a new DNA sequence (shown as swSeq ID Nol), which was stored in the PBS plasmid (pBS-thrS). There was a 2156bp from 53bp to 2209bp. ORF, which encodes a new protein (as shown in Seq ID No 2). We named this protein threonine transfer RNA synthetase. Example 2: Expression and purification of threonyltransferase synthetase protein in E. coli system

Based on the corresponding gene sequence of the new human threonine transfer ribonucleic acid synthetase protein, a pair of specific amplification primers were designed with the following sequence: Primer 1: 5, GGGAGAAGCGGCGATAATCTG 3 '

Primer 2: 5 'GTTTGTATTTATTTATTTATTTATT 3'

These two sequences contain Ndel and Hindlll digestion sites, respectively, followed by the coding sequences of the target gene 3, 5 and 5, respectively, using the pBS plasmid containing the full-length target gene as a template for the PCR reaction. The restriction sites of Ndel and Hindlll correspond to the selective endonuclease sites on the expression vector plasmid PTSA-18. Ndel and Hindlll were used to digest and ligate the amplified sequence and plasmid PTSA-18, respectively. The recombinant plasmid was transformed into the host strain E. coli BL21 (DE3) plySs, and induced by IPTG for expression. The expressed product was sonicated and heat-denatured, and then applied to a DEAE column to obtain a purified target protein. Example 3: Homologous search of cDNA clones

The novel human threonyl transfer ribonucleic acid synthetase protein polynucleotide sequence provided by the present invention and its encoded protein sequence are used to perform homology search in databases such as Genbank and Swissport. The program for searching is called Blast (Basic local Alignment search tool) (1993 Proc Nat Acad Sci 90: 5873- 5877), Blast can find many genes that are homologous to threonyl transfer ribonucleic acid synthetase protein, among which the gene with the most homology to the gene we invented, Its encoded protein has the accession number M63180 in Genbank. These retrieved genes 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. Functional prediction of new proteins can be analyzed using the Motif program. The results of the homology search are shown below. The results show that the human threonyl transfer ribonucleic acid synthetase protein provided by the present invention has 61% homology with the human threonyl transfer ribonucleic acid synthetase protein provided in the Genband database ( (See the table below for details). -61-

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S, Z00 / 00ND / lDd 66661/10 OM Example 4: Production of anti-threonyl transfer ribonuclease synthetase antibody The following threonyl transfer ribonucleic acid-synthetic peptide was synthesized using a peptide synthesizer (PE-ABI): H2-Leu Tyr Gin Arg Trp Arg Cys Leu Arg Leu-COOH. The polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively. For the method, see: Avrameas, et al. 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 μ ₈ / π1 bovine serum albumin peptide complex was used as an ELISA to determine the antibody titer in rabbit serum. Total _Ig G was isolated from antibody-positive rabbit serum using protein A-Sepharose. The peptide was bound to a cyanogen bromide-activated Sepharose 4B column, and the anti-peptide antibody was separated from the total IgG by affinity chromatography. The immunoprecipitation method proved that the purified antibody could specifically bind to threonyltransferase synthetase.

Sequence Listing

(1) General information:

 (i) The applicant:

 (A) Name: Shanghai Shengyuan Gene Development Co., Ltd.

 (B) Street: Room 610, 668 East Beijing Road

 (C) City: Shanghai

 (E) Country: China

 (F) Postal code: 200001

 (ii) Title of invention: Gene encoding a novel threonyl transfer ribonucleic acid synthetase, and application and preparation method thereof

 (iii) Number of sequences: 2

 (1) General information:

 (2) Information of SEQ ID NO: 1:

 (i) Sequence characteristics:

 (A) Length: 2741bp

 (B) Type: Nucleic acid

 (C) Chain: double strand

 (D) Topological structure: linear

 (ii) Molecular type: cDNA

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

 1 GGGAGAAGCGGCGATAATCTGTTTGAGGATGTAGGCACTGGTGTGAAGGAACATGGCCCT 61 GTATCAGAGGTGGCGGTGTCCGGCTCTCCAAGGTTTACAGGCTTGCAGGCTACACACGGC 121 AGTTGTGTCGACCCCTCCACGCTGGTTGGCAAGAGGGGGGTCGCGGGGTT

241 ATCACTTCCTGGAGGCCAGAAAATTGATGCTGTGGCATGGAACACAACCCCCTACCAACT 301 AGCCCGGCAGATCAGTTCAACACTGGCAGATACTGCAGTGGCTGCTCAAGTGAATGGAGA

361 ACCTTATGATCTGGAGCGGCCCTTGGAGACAGATTCTGACCTCAGATTTCTGACATTCGA

421 TTCCCCAGAGGGGAAAGCAGTGTTCTGGCACTCCAGCACCCATGTCCTGGGGGCAGCAGC

481 TGAACAATTCCTAGGTGCTGTTCTCTGCAGAGGTCCAAGTACAGAATATGGCTTTTACCA

541 TGATTTCTTCCTGGGAAAGGAGAGGACAATCCGGGGCTCAGAGCTGCCTGTTTTGGAGCG

601 GATTTGCCAGGAACTTACAGCTGCTGCTCGACCCTTCCGGAGGCTAGAGGCTTCACGGGA

661 TCAGCTTCGCCAGTTGTTCAAGGATAACCCCTTTAAGCTTCACTTGATTGAGGAGAAAGT

721 GACAGGTCCAACAGCAACAGTATATGGGTGTGGCACATTGGTTGACCTTTGCCAGGGCCC

781 CCACCTTCGGCATACTGGACAGATTGGAGGACTGAAGCTGCTATCGAACTCATCATCCTT

841 ATGGAGGTCTTCAGGGGCCCCAGAGACACTGCAGAGAGTGTCAGGGATTTCCTTCCCTAC

901 AACAGAATTGCTGAGGGTCTGGGAAGCATGGAGGGAGGAAGCAGAATTGCGGGACCACCG

961 GCGCATTGGGAAGGAACAGGAGCTCTTCTTCTTCCATGAACTGAGCCCTGGGAGCTGCTT

1021 CTTCCTGCCACGAGGGACAAGGGTGTATAATGCACTAGTGGCGTTTATCAGGGCTGAGTA

1081 TGCCCATCGTGGTTTCTCCGAGGTGAAAACTCCCACACTGTTTTCTACGAAGCTCTGGGA

1141 ACAGTCAGGGCACTGGGAGCATTATCAGGAAGACATGTTTGCCGTGCAGCCCCCAGGCTC

1201 TGACAGGCCTCCCAGCTCCCAGAGTGACGATTCTACCAGGCATATCACAGATACACTCGC

1261 CCTCAAGCCTATGAACTGCCCTGCACACTGCCTGATGTTCGCCCACCGGCCCAGATCCTG

1321 GCGGGAACTGCCCCTGCGACTAGCTGACTTTGGGGCTCTACACCGGGCCGAAGCCTCTGG

1381 TGGTCTGGGGGGACTGACCCGACTGCGGTGCTTCCAGCAGGATGACGCTCACATCTTCTG

1441 TACAACAGATCAGCTGGAAGCAGAGATCCAAAGCTGTCTTGATTTCCTCCGTTCCGTCTA

1501 TGCCGTTCTTGGCTTCTCCTTCCGCCTGGCACTGTCCACCCGGCCATCTGGCTTCCTGGG

1561 GGACCCTTGCCTTTGGGACCAGGCCGAACAGGTCCTTAAACAGGCCCTGAAGGAATTTGG

1621 AGAACCCTGGGACCTCAACTCTGGAGATGGTGCCTTCTATGGACCTAAGATTGACGTGCA

1681 CCTCCACGATGCCCTGGGCCGGCCACATCAGTGTGGGACAATTCAGCTTGACTTCCAACT

1741 GCCCCTGAGATTTGACCTCCAGTATAAGGGGCAGGCGGGTGCCCTGGAGCGTCCAGTCCT

1801 CATTCACCGAGCAGTGCTCGGTTCTGTGGAAAGACTGTTGGGAGTGCTGGCAGAAAGCTG 1861 CGGGGGGAAATGGCCACTGTGGCTGTCCCCGTTCCAGGTGGTGGTCATCCCTGTGGGGAG 1921 TGAGCAAGAGGAATACGCCAAAGAGGCACAGCAGAGCCTGCGGGCTGCAGGACTGGTCAG 1981 TGACCTGGATGCAGACTCTGGACTGACCCTCAGCCGGAGAATCCGCCGGGCCCAGCTTGC 2041 CCACTACAATTTTCAGTTTGTGGTTGGCCAGAAAGAGCAAAGTAAGAGAACAGTGAACAT 2101 TCGGACTCGAGATAATCGTCGCCTTGGGGAGTGGGACTTGCCTGAGGCTGTGCAGCGACT 2161 GGTGGAGCTACAGAACACGAGGGTCCCAAATGCCGAAGAAATTTTCTGAGCCTTTGTACA 2221 TAGATGAGGCAAAAACCTGCGAGTGCCATCAGCCTCCCTCACATGGGAGACCCCAACCCA 2281 GCTGACAATGTGGAGCCCCCAGAACTTCAGAACTGTGTGGAGGCACATGTCTGCTCTCCT 2341 GAAAAGAGACTTGGTTTGGGGACCCCACAAAAGGAGGGAAGCTGTAGCTGTTTGGATGTG 2401 AGGAGAATGAAACTACAAAAAAAATAAATTGGGCCAGGCGCAGTGGCTCATGCCTGTAAT 2461 CCCAGCACTCTGGGAGGCTGAGGCGGACGGATCATGAGGTCAGGAGATCAAGACCACCCT 2521 GGCTAACACGGTGAAACCCTGTCTCTACTAAAAATACAAAAAATTAGCCGGGCATGGTGG 2581 CACACGCCTGTAATCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATCGCTTGAACCCGGG 2641 AGGTAGAGGTTGCAGTGAGCTGAGATTGCGCCACTGCACCCCCCTAGGCGACAGAGCGAG 2701 ACTCTGTCTCTAAATAAATAAATAAATAAATAAATACAAAC

 (2) Information of SEQ ID NO: 2:

 (i) Sequence characteristics:

 (A) Length: 718 amino acids

 (B) Type: Amino acid

 (D) Topological structure: linear

 (ii) Molecular type: peptide

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

 1 Met Ala Leu Tyr Gin Arg Trp Arg Cys Leu Arg Leu Gin Gly Leu

16 Gin Ala Cys Arg Leu His Thr Ala Val Val Ser Thr Pro Pro Arg

31 Trp Leu Ala Glu Arg Leu Gly Leu Phe Glu Glu Leu Trp Ala Ala

46 Gin Val Lys Arg Leu Ala Ser Met Ala Gin Lys Glu Pro Arg Thr

61 lie Lys lie Ser Leu Pro Gly Gly Gin Lys lie Asp Ala Val Ala

76 Trp Asn Thr Thr Pro Tyr Gin Leu Ala Arg Gin lie Ser Ser Thr

91 Leu Ala Asp Thr Ala Val Ala Ala Gin Val Asn Gly Glu Pro Tyr . · ¾ ΐ¾ "TO na 90Z

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Claims

Rights request 1. An isolated threonine transfer ribonucleic acid synthesis reagent comprising a polypeptide having the amino acid sequence shown in SEQ ID No. 2, or a conservative variant polypeptide thereof, or an active fragment thereof, or an active derivative thereof.  The polypeptide according to claim 1, which is a polypeptide having the amino acid sequence of SEQ ID No. 2.  3. An isolated polynucleotide comprising a nucleotide sequence that is at least 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 sequence having positions 53-2209 in SEQ ID No. 1;  (b) A sequence having positions 1 to 2741 in SEQ ID No. 1.  A recombinant vector comprising the polynucleotide sequence according to claim 3, 4 or 5.  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, 4 or 5. 8. A method for preparing a polypeptide having human threonine transfer ribonucleic acid synthetase activity, comprising: (a) culturing the host cell according to claim 7 under conditions suitable for expression of threonyl transfer ribonucleic acid synthesis;  (b) A polypeptide having threonyltransferase synthetase activity is isolated from the culture.  An antibody capable of specifically binding to a threonyl transfer ribonucleic acid synthetase according to claim 1.  10. A method for screening a compound that mimics, promotes, antagonizes, or inhibits threonine-transferred ribonucleic acid-synthesizing alcohol activity, comprising using the polypeptide of claim 1.  The compound obtained by the method according to claim 10, which has an activity that mimics, promotes, antagonizes or inhibits the threonyl transfer ribonucleic acid synthesizing alcohol activity of claim 1.  A method for regulating the activity of threonyl transfer ribonucleic acid synthase in vivo and in vitro by using the compound according to claim 11.  13. A method for detecting a disease or susceptibility to a disease related to a polypeptide according to claim 1 or 2, characterized by detecting an abnormality in the expression amount and / or activity of the polypeptide according to claim 1, or detecting the cause of the claim The abnormal expression level and / or activity of the polypeptide of 1 encodes a mutation in the nucleotide sequence of the polypeptide of claim 1.  14. The use of a polypeptide according to claim 1 or 2 for screening an agonist that promotes threonyl transfer ribonucleic acid synthesizing alcohol activity, or an antagonist that inhibits threonyl transfer ribonucleic acid synthase activity, It is used for peptide fingerprint identification.  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 in the preparation of a pharmaceutical composition for the treatment of a disease associated with abnormal activity of threonine transfer ribonucleic acid synthase. 17. The use according to claim 16, wherein the disease is a disease caused by low or loss of threonyltransferase synthetase function, including various cancers.