WO2001031024A1 - Nouveau polypeptide, threonine synthetase 71, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, threonine synthetase 71, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001031024A1
WO2001031024A1 PCT/CN2000/000396 CN0000396W WO0131024A1 WO 2001031024 A1 WO2001031024 A1 WO 2001031024A1 CN 0000396 W CN0000396 W CN 0000396W WO 0131024 A1 WO0131024 A1 WO 0131024A1
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polypeptide
polynucleotide
threonine synthase
seq
threonine
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PCT/CN2000/000396
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English (en)
Chinese (zh)
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Yumin Mao
Yi Xie
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Shanghai Bio Road Gene Development Ltd.
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Priority to AU13785/01A priority Critical patent/AU1378501A/en
Publication of WO2001031024A1 publication Critical patent/WO2001031024A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, threonine synthase 71, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a method and application for preparing the polynucleotide and polypeptide.
  • the branch pathway from aspartic acid to threonine involves five steps and requires the catalysis of three enzymes. Among them, threonine synthetase catalyzes the final step, which is the transfer of 0-phosphate-L-homoserine to threonine and inorganic phosphorus.
  • This enzyme is a 5'-pyridoxal phosphate-dependent enzyme, which includes a complete open reading frame (encoding 428 codes) containing 1542 nucleotides, and two partial reading frames (one after 273bp upstream of the start code). 630 bp reading frame, and the other is a 277 bp reading frame located 84 bp downstream of the stop code) (Regu la Al tmann-Joh l. Peter Phi li ppsen 1996 Mo l Gen Gene t 250: 69-80) . A TATA box and a GCN4 protein binding site are present in the 5 'non-coding region.
  • the GCN4 protein binding site is a small repeat of 5'-TGACTC-3 'in the 5' non-coding region.
  • This protein is a general regulatory protein for amino acid synthesis. 21 bp downstream of the stop code, there is a GC-rich dichotomous symmetry region. During transcription, this region can form a stable stem region and secondary structure of the salamander. This structure is independent of the P-factor termination site and may be a terminator transcribed at the end of the operon. Transcription termination signals may also be present in some regions that meet the following conditions: 1) AT-rich regions located in the transcription termination region; 2) CAATCAA-related, and 3) the dimeric symmetric region precedes the region.
  • the cistron region of threonine synthetase is composed of a long bimeric symmetric sequence. This region may be the termination or antecedent site of transcription (Rosenberg, M. and Court, D. 1979 Ann. Rev. Genet. 13, 319-353)
  • Threonine synthase contains a conserved pyridoxal phosphate site, the gene sequence of this site is PE] -X- [STVG] -X- [AS]-[FYI] -K- [DLISA]-[RV F]-[GA]-[LIVMGA], (Parsot C1986 EMBO] 5: 3013-3019)
  • the polypeptides of the present inventors have high homology with threonine synthetase. Threonine synthase
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a threonine synthetase 7 1.
  • Another object of the present invention is to provide a genetically engineered host cell comprising a polynucleotide encoding a threonine synthetase 71.
  • Another object of the present invention is to provide a method for producing threonine synthase 71.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention, threonine synthase 71.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors against the polypeptide of the present invention, threonine synthase 71.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities in threonine synthase 71.
  • a novel isolated threonine synthetase 71 is provided.
  • the polypeptide is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID NO: 2, or a conservative variant polypeptide thereof, or Its active fragment, or its active derivative, analog.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • a polynucleotide encoding these isolated polypeptides, the polynucleotide comprising a nucleotide sequence having at least 7 nucleotides with a nucleotide sequence selected from the group consisting of 0% identity: (a) a polynucleotide encoding the aforementioned threonine synthetase 7 1; (b) a polynucleotide complementary to the polynucleotide (a).
  • the polynucleotide encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 2.
  • the sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 64-2007 in SEQ ID NO: 1; and (b) a sequence having positions 1-2 in SEQ ID NO: 1 066-bit sequence.
  • a vector containing the above polynucleotide, and a host cell transformed or transduced by the vector or a host cell directly transformed or transduced by the above polynucleotide are provided.
  • Fig. 1 is a comparison diagram of the amino acid sequence homology of the threonine synthetase 71 of the present invention and the threonine synthetase of Helicobacter.
  • the upper sequence is threonine synthase 71 and the lower sequence is threonine synthase.
  • Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+”.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated threonine synthase 71.
  • 7 1 kDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • 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).
  • 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 in the natural state .
  • isolated threonine synthase 71 means that threonine synthase 71 is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify threonine synthase 71 using standard protein purification techniques. Substantially pure polypeptides produce a single main band on a non-reducing polyacrylamide gel. The purity of the threonine synthase 71 peptide can be analyzed by amino acid sequence.
  • the present invention provides a novel polypeptide, threonine synthase 71, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention may be naturally purified products or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of threonine synthase 7 1.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the threonine synthase 71 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or ( ⁇ ⁇ )
  • Such a polypeptide sequence in which the mature polypeptide is fused with another compound such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol
  • a polypeptide sequence in which an additional amino acid sequence is fused into the mature polypeptide (Such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protease sequence)
  • such fragments, derivatives, and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence with a total length of 2066 bases, and its open reading frame (64-2007) encodes 647 amino acids. According to the amino acid sequence homology comparison, it was found that this polypeptide has 36% homology with the threonine synthetase of Helicobacter. It can be deduced that the threonine synthase 71 has a similar structure and Features.
  • 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.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide is meant to include polynucleotides that encode such polypeptides and polynucleotides that include additional coding and / or noncoding sequences.
  • the present invention also relates to a variant of the polynucleotide described above, which encodes the same amino group as the present invention. Acid sequences of polypeptides or fragments, analogs and derivatives of polypeptides. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity, between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) Add a denaturant during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% F i co ll, 42 ° C, etc .; or (3) only between the two sequences Crosses occur at least 95% or more, and more preferably 97% or more.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, most preferably at least 100 More than nucleotides.
  • Nucleic acid fragments can also be used in nucleic acid amplification techniques such as PCR to identify and / or isolate polynucleotides encoding threonine synthase 71.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • the specific polynucleotide sequence encoding the threonine synthetase 71 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: v Hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleotides with common structural characteristics Fragment.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the CDM of interest is to isolate mRM from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • Q i agene There are many mature techniques for mRNA extraction, and kits are also commercially available (Q i agene).
  • 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): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) measuring the level of the transcript of threonine synthase 71; (4) ) Detection of protein products expressed by genes through immunological techniques or determination of biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is usually a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product expressed by the threonine synthase 71 gene.
  • ELISA enzyme-linked immunosorbent assay
  • a method using PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-rapid cDNA end rapid amplification method
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be measured by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising a polynucleotide of the present invention, and a host cell that is genetically engineered using the vector of the present invention or directly using a threonine synthase 71 coding sequence, and a recombinant technology for producing a polypeptide of the present invention method.
  • the polynucleotide sequence encoding the threonine synthase 71 can be inserted into a vector to construct o 01 31024 pcT / o ⁇ 003 % into a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding threonine synthase 71 and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Mol ecular Cloning, A Laboratory Manua, Cold Spruing Harbor Labora tory. New York, 1989) Efficiently 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.
  • Expression Vectors also include ribosome binding sites and transcription terminators for translation initiation. Inserting enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting effects of DNA expression Factors, usually about 10 to 300 base pairs, act on promoters to enhance gene transcription. Examples include at the origin of replication 100 to 270 base pairs of the side of the SV40 enhancer, adenovirus enhancers, and the like enhancer on the late side of the replication origin polyoma.
  • 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.
  • 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.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding a threonine synthetase 71 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetic engineering containing the polynucleotide or the recombinant vector.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: E. coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as fly S 2 or Sf 9; animal cells such as CH0, COS or Bowes s melanoma cells Wait.
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of DNA uptake can be harvested after exponential growth phase, with (: Treatment 1 2, steps well known in the art with alternative is MgC l 2
  • transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and lipid Body packaging, etc.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant threonine synthetase 71 (Science, 1 984; 224: 14 31) by conventional recombinant DNA technology. Generally there are the following steps:
  • 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.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid
  • the polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection and immune diseases.
  • the abnormal expression of the polypeptide of the present invention will affect the synthesis of threonine. Because threonine is an indispensable part of the amino acid metabolism pathway, it is also one of the indispensable amino acids in the human body. Therefore, abnormal synthesis of threonine will cause physiological and metabolic disorders, and then affect many systemic functional disorders. .
  • the abnormal expression of the polypeptide of the present invention can cause, but is not limited to, the following diseases: thromboembolism, homocystinuria, excessive limb length, chest depression, lens ectopic, spontaneous pneumothorax, osteoporosis, mental retardation and mental disorders.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) threonine synthase 71.
  • Agonists enhance biological functions such as threonine synthase 71 to stimulate cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing threonine synthase 71 can be cultured together with labeled threonine synthase 71 in the presence of drugs. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of threonine synthase 71 include antibodies, compounds, receptor deletions, and analogs that have been screened.
  • An antagonist of threonine synthase 71 can bind to threonine synthase 71 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions .
  • threonine synthase 71 When screening compounds as antagonists, threonine synthase 71 can be added to the bioanalytical assay to determine whether the compound is an antagonist by measuring the effect of the compound on the interaction between threonine synthase 71 and its receptor. . Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to threonine synthase 71 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. During screening, the threonine synthase 71 molecule should generally be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies directed against the threonine synthase 71 epitope. These antibodies include (but are not limited to): Doklon antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, and fragments from Fab expression libraries.
  • Polyclonal antibody production can be obtained by direct injection of threonine synthase 71 into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant. Wait.
  • Techniques for preparing monoclonal antibodies to threonine synthase 7 1 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 271: 495-497), three tumor technology, human B-cell hybridoma technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions to non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851). 0 Existing techniques for producing single-chain antibodies (US Pa t No. 4946778) can also be used to produce single chain antibodies against threonine synthase 71.
  • Anti-threonine synthase 71 antibodies can be used in immunohistochemistry to detect threonine synthase 71 in biopsy specimens.
  • Monoclonal antibodies that bind to threonine synthase 71 can also be labeled with radioisotopes and injected into the body to track their location and distribution.
  • This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • threonine synthase 71 high affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill threonine synthase 71 positive cells .
  • the antibodies of the present invention can be used to treat or prevent diseases related to threonine synthase 71.
  • Administration of an appropriate dose of the antibody can stimulate or block the production or activity of threonine synthase 71.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of threonine synthase 71 levels. These tests are well known in the art and include FISH assays and radioimmunoassays. The level of threonine synthase 71 detected in the test can be used to explain the importance of threonine synthase 71 in various diseases and to diagnose diseases in which threonine synthase 71 functions.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • the polynucleotide encoding threonine synthase 71 can also be used for a variety of therapeutic purposes. Gene therapy technology can be used to treat abnormal cell proliferation, development, or metabolism caused by the non-expression or abnormal / inactive expression of threonine synthase 71.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated threonine synthase 71 to inhibit endogenous threonine synthase 71 activity.
  • a variant threonine synthase 71 may be a shortened threonine synthase 71 lacking a signaling domain, and although it can bind to a downstream substrate, it lacks signaling activity.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, and the like can be used to transfer a polynucleotide encoding threonine synthase 71 into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a threonine synthase 71 can be found in the existing literature (Sambrook, etal.).
  • the recombinant polynucleotide encoding threonine synthase 71 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit threonine synthase 71 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that specifically decomposes 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 using any existing RNA or DNA synthesis technology, such as solid-phase phosphate amide chemical synthesis to synthesize oligonucleotides.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of the D sequence encoding the RNA.
  • This DNA sequence has been integrated downstream of the RNA polymerase promoter of the vector.
  • it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphorothioate or peptide bonds instead of phosphodiester bonds.
  • a polynucleotide encoding threonine synthase 71 can be used for the diagnosis of diseases related to threonine synthase 71.
  • a polynucleotide encoding threonine synthase 71 can be used to detect the expression of threonine synthase 71 or the abnormal expression of threonine synthase 71 in a disease state.
  • the DM sequence encoding threonine synthase 71 can be used to hybridize biopsy specimens to determine the expression status of threonine synthase 71.
  • Hybridization techniques include Sout hern blotting, Nor thern blotting, in situ hybridization, and the like.
  • RNA-polymerase chain reaction RNA-polymerase chain reaction
  • Threonine Synthase 71 Mutated Form Includes DNA Sequence with Normal Wild Type Threonine Synthase 71 Compared to point mutations, translocations, deletions, recombinations, and any other abnormalities. 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 blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for marking chromosome positions.
  • an important first step is to locate these DNA sequences on a chromosome.
  • a PCR primer (preferably 15-35bp) is prepared from the cDNA, and the sequence can be located on the chromosome. 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.
  • 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 chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones to metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • 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, Mendel ian Inheritance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • cDNA or genomic sequences between the affected and unaffected individuals need to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable using cDNA sequence-based PCR. Accurately located based on the resolution capabilities of current physical mapping and gene mapping technologies
  • the cDNA to a chromosomal region associated with a disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution and one gene per 20 kb).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Threonine synthase 71 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dose range of threonine synthase 71 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.
  • the sequence of the threonine synthase 71 of the present invention and the protein sequence encoded by the threonine synthetase 71 were coded using the Blast program (Basic local alignment search tool) [Altschul, SF et al. J. Mol. Biol. 1990; 215: 403-10] Perform homology search in Genbank, Swissport and other databases.
  • the gene with the highest homology to the threonine synthetase 71 of the present invention is a known threonine synthase of Helicobacter, and its encoded protein has the accession number of AE001448 in Genbank.
  • the results of protein homology are shown in Figure 1. The two are highly homologous, with an identity of 36% and a similarity of 55%.
  • Example 3 Cloning of a gene encoding threonine synthase 71 by RT-PCR
  • CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer for reverse transcription reaction. After purification using Qiagene's kit, the following primers were used for PCR amplification:
  • Primerl 5,-GGTCAGAAACTAGGTTGTTGCGTC-3 '(SEQ ID NO: 3)
  • Primer2 5'- AGTTTGAGATTTATTATTGCATGC-3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp of the 5th end of SEQ ID NO: 1;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification conditions 50 ⁇ l / L KC1, 10mmol / L Tris in a reaction volume of 50 ⁇ 1-
  • This method involves acid guanidinium thiocyanate phenol-chloroform extraction. I.e. with 4M guanidinium isothiocyanate -25mM sodium citrate, 0.2M sodium acetate (P H4.0) of the tissue was homogenized phenol, 1 volume and 1/5 volume of chloroform - isoamyl alcohol (49: 1), centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The obtained RM precipitate was washed with 70% ethanol, dried and dissolved in water.
  • a 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RM was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 (pH7.4) -5 x SSC-5 x Denhardt; s solution and 200 ⁇ g / ml salmon sperm DNA. After hybridization, place the filter at 1 x SSC-0.1 ° /. Wash in SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 5 In vitro expression, isolation and purification of recombinant threonine synthase 71
  • Primer3 5'- CATCCATGGATGAGTGTGTCTGAAAAATTACAGG-3 '(Seq ID No: 5)
  • Primer4 5'- CCCGAGCTCCCTATGAATTGATTTTGGACAAGTrG-3 '(Seq ID No: 6)
  • the 5' ends of these two primers contain Ncol and Sacl digestion sites, respectively, followed by the coding sequences of the 5 'and 3' ends of the target gene, respectively.
  • Ncol and Sacl restriction sites correspond to selective endonuclease sites on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3).
  • the 0172f08 plasmid was used as a template for the PCR reaction.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of P BS-0172f 08 plasmid, primers Primer-3 and Primer-4 points, and 'J is lOpmol, Advantage polymerase Mix
  • the cells were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. Chromatography was performed using an His. Bind Quick Cartridge (product of Novageri) that can bind 6 histidines (6His-Tag).
  • the purified protein threonine synthase 71 was obtained. After SDS-PAGE electrophoresis, a single band was obtained at 71 kDa ( Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by Edams hydrolysis method. As a result, the 15 amino acids at the N-terminus were identical to the 15 amino acid residues at the N-terminus shown in SEQ ID NO: 2.
  • peptide synthesizer product of PE company
  • NH 2 -Me t-Ser-Val-Ser-Gl u-Lys-Leu-G 1 n-Asp-Va 1 -G 1 yA s nG 1 uG 1 n-Phe- C00H SEQ ID NO: 7
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • Rabbits were immunized with 4 mg of the above-mentioned jk cyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost the immunity once.
  • a titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum.
  • Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose.
  • the peptide was bound to a cyanogen bromide-activated Se P ha r0Se 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 threonine synthase 71.

Abstract

L'invention concerne un nouveau polypeptide, une thréonine synthétase 71, et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la thréonine synthétase 71.
PCT/CN2000/000396 1999-10-27 2000-10-27 Nouveau polypeptide, threonine synthetase 71, et polynucleotide codant pour ce polypeptide WO2001031024A1 (fr)

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CN99119868.9 1999-10-27
CN 99119868 CN1303944A (zh) 1999-10-27 1999-10-27 一种新的多肽——苏氨酸合成酶71和编码这种多肽的多核苷酸

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6806060B2 (en) 2001-12-07 2004-10-19 Icoria, Inc. Methods for the identification of inhibitors of threonine synthase as antibiotics

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63237793A (ja) * 1987-03-26 1988-10-04 Ajinomoto Co Inc Ｌ−スレオニンの製造法
DD262040A1 (de) * 1987-07-13 1988-11-16 Adw Ddr Verfahren zur gewinnung von hefe-rezipientenstaemmen der gattung saccharomyces
WO1988009819A2 (fr) * 1987-06-12 1988-12-15 Massachusetts Institute Of Technology Passage biosynthetique pour threonine de c. glutamicum

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63237793A (ja) * 1987-03-26 1988-10-04 Ajinomoto Co Inc Ｌ−スレオニンの製造法
WO1988009819A2 (fr) * 1987-06-12 1988-12-15 Massachusetts Institute Of Technology Passage biosynthetique pour threonine de c. glutamicum
DD262040A1 (de) * 1987-07-13 1988-11-16 Adw Ddr Verfahren zur gewinnung von hefe-rezipientenstaemmen der gattung saccharomyces

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6806060B2 (en) 2001-12-07 2004-10-19 Icoria, Inc. Methods for the identification of inhibitors of threonine synthase as antibiotics

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AU1378501A (en) 2001-05-08

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