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WO2001031001A1 - Nouveau polypeptide, facteur auxiliaire 28 du facteur de demarrage de la traduction, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, facteur auxiliaire 28 du facteur de demarrage de la traduction, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001031001A1
WO2001031001A1 PCT/CN2000/000382 CN0000382W WO0131001A1 WO 2001031001 A1 WO2001031001 A1 WO 2001031001A1 CN 0000382 W CN0000382 W CN 0000382W WO 0131001 A1 WO0131001 A1 WO 0131001A1
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Prior art keywords
polypeptide
polynucleotide
translation initiation
initiation factor
factor cofactor
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Bioroad Gene Development Ltd
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Shanghai Bioroad Gene Development Ltd
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Priority to AU12646/01A priority Critical patent/AU1264601A/en
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Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • 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 new polypeptide ⁇ M translation initiation factor cofactor 28, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a method and application for preparing such polynucleotides and polypeptides.
  • Protein biosynthesis is the translation process of genetic information. The translation process requires mRNA, ribosome, various amino acids, tRNA, and the participation of various protein factors, including initiation factors, elongation factors, and termination factors.
  • elF such as eIF1, eIF2, eIF3, eIF4, etc. are eukaryotic biosynthesis initiation factors.
  • Met-tRNA, ⁇ eIF-2 ⁇ GTP ternary complex is the first step in the initiation process of eukaryotic protein synthesis.
  • the second step is the combination of ⁇ ⁇ 1411 ⁇ 1 and the 40S ribosomal subunit.
  • Me t-tRM! ⁇ 40S. MRNA complex is formed, and the above two steps are regulated by several helper protein factors (Gupta eta l. 1993; Her shey 1991; Merrick and Hershey 1996).
  • GTP binds to eIF-2 to form a binary complex, and then binds to Met-tRNA! Raet .
  • GTP binds to elF-2 to enhance the affinity of eIF-2 to Met-tRNA! Net , a cofactor eIF2C enhances the stability of the ternary complex and prevents its decomposition.
  • the cofactor eIF2C also stabilizes Met-tRNA!. 40S ⁇ mRNA complex (Roy et al., 1988).
  • eIF2C was first isolated from rabbit reticulocyte lysin with a molecular weight of 140KD. It is easily degraded. The three degrading peptides are 94KD, 50KD, and 25KD.
  • the open reading frame sequence of eIF2C gene decodes 813 amino acids. Its starting AUG is located in the translated strong signal sequence (CGAGAUGG). It contains purines at the -3 and +4 positions. No polyadenosine was found in the non-coding region at the 3 'end. acid.
  • the total GC content of the cloned eIF2CcDNA accounted for 60%, which was relatively lower than some human translation initiation factors eIF-2 cc (GC 40%), eIF-2 ⁇ (GC 42%), and eIF-2 V (GC 43%). high.
  • the eIF2C gene open reading frame sequence decoded peptide was 90KD, and its molecular weight was different from that of 140KDeIF2C in rabbit reticinolysin suggesting post-translational modifications. Two N-glycosylation sites, two Amidation site, eight N-tetradecanylation sites, multiple phosphorylation sites.
  • the 94KD peptide purified from rabbit retinolysin is a glycosylated protein with a high isoelectric point and rich in charged amino acids (Cheng zou et al., 1998).
  • the human translation initiation factor cofactor 28 gene of the present invention has 80% homology with the rabbit eIF2C 90 gene at the protein level (homologous protein number AF005355), and its domain is similar to the characteristic structure of the eIF2C gene family Domain 1-High GC content, post-translational modification, possible N-glycosylation sites, amidation sites, N-tetradecanylation sites, and multiple phosphorylation sites, based on the above Therefore, the novel gene of the present invention is considered to be a gene encoding a human translation initiation factor cofactor gene family, named as human translation initiation factor cofactor 28 ′, and from this it is deduced that its domain is similar to the eIF2C gene family domain, and has Similar biological functions.
  • EIF2C was also found in rabbit kidney cDNA, suggesting that the eIF2C gene is a simple repeat in the rabbit chromosome (Cheng zou et al., 1998).
  • Some studies have shown that eIF2C-like activity is widely present in eukaryotic tissues, such as mouse ascites tumor cells, wheat germ, yeast, etc. (Chakravarty et al., 1985; Dasgupta et al., 1978; Osterhout et al., 1983; Ahmad et al 1985).
  • 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 translation initiation factor cofactor 28.
  • Another object of the invention is to provide a genetically engineered host cell containing a polynucleotide encoding a translation initiation factor cofactor 28.
  • Another object of the present invention is to provide a method for producing a translation initiation cofactor 28.
  • Another object of the present invention is to provide antibodies against the polypeptide-translation initiation factor cofactor 28 of the present invention.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide-translation initiation factor cofactor 28 of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities of translation initiation factor cofactor 28.
  • Summary of invention In a first aspect of the present invention, a novel isolated translation initiation factor cofactor 28 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, the nucleotide sequence and at least 80% of the nucleotide sequence selected from the group Identities: (a) a polynucleotide encoding the aforementioned translation initiation factor cofactor 28; (b) a polynucleotide complementary to the polynucleotide (a).
  • the polynucleotide encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 2.
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 878-1636 in SEQ ID NO: 1; and (b) a sequence having 1-2951 in SEQ ID NO: 1 Sequence of bits.
  • Fig. 1 is a comparison diagram of amino acid sequence homology of translation initiation factor cofactor 28 of the present invention and rabbit transcription initiation factor eF2C.
  • the upper sequence is the translation initiation factor cofactor 28, and the lower sequence is the rabbit transcription initiation factor e lF2C.
  • 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 translation initiation factor cofactor 28.
  • 28kDa 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 translation initiation factor cofactor 28 means translation initiation factor helper Factor 28 is essentially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify the translation initiation factor cofactor 28 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the translation initiation factor cofactor 28 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide-translation initiation factor cofactor 28, which basically consists of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptide of the present invention may be a naturally purified product or a chemically synthesized product, or may be produced from a prokaryotic or eukaryotic host (for example, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant technology. 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 the translation initiation factor cofactor 28.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the translation initiation factor cofactor 28 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 2951 bases, and its open reading frame (878-1636) encodes 252 amino acids. According to the amino acid sequence homology comparison, it was found that this polypeptide has 80% homology with rabbit's transcription initiation factor e lF2C. It can be inferred that the translation initiation factor cofactor 28 has rabbit's transcription initiation The factor e lF2C has a similar structure and function.
  • the polynucleotide of the present invention may be in the D form or the RNA form.
  • DNA forms include cD, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DM can be coded or non-coded.
  • 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 refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • 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 such as 50 when hybridizing.
  • 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, and most preferably at least 100 nuclei. Glycylic acid or more. Nucleic acid fragments can also be used for nucleic acid amplification Amplification techniques (eg, PCR) to identify and / or isolate a polynucleotide encoding a translation initiation factor cofactor 28.
  • the polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • polynucleotide sequence encoding the translation initiation factor cofactor 28 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: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) separating the double-stranded DM 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 cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Moleculolar Cloning, A Labora tory Manua, Collspring Harbor Labora tory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • 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-DM or DM-RNA hybridization; (2) the presence or absence of a marker gene function; (3) measuring the level of the transcript of the translation initiation factor cofactor 28; ( 4) Detecting gene-expressed protein products by immunological techniques or by measuring 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).
  • the protein product of the translation initiation factor cofactor 28 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • Method for Amplifying DNA / RNA by PCR are preferred for obtaining the genes of the invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • 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 / R fragment 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 the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a translation initiation factor cofactor 28 coding sequence, and the recombinant technology to produce the polypeptide of the present invention Methods.
  • a polynucleotide sequence encoding a translation initiation factor cofactor 28 may be inserted into a vector to constitute 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 an expression vector is that it usually contains a replication origin, a promoter, a marker gene, and translation control elements. Methods well known to those skilled in the art can be used to construct a DNA sequence containing a translation initiation factor cofactor 28 and a suitable Expression vector for transcriptional / translational regulatory elements.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for D expression, 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.
  • 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 translation initiation factor cofactor 28 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • 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.
  • Escherichia coli, Streptomyces bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells insect cells
  • fly S2 or Sf 9 animal cells
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • 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 absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • polynucleotide sequence of the present invention can be used to express or produce a recombinant translation initiation factor cofactor 28 (Science, 1984; 224: 1431). 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
  • polypeptides of the present invention as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.
  • the activity of the translation initiation cofactor eIF2C is widely present in eukaryotic tissues, such as rabbit reticulum, rabbit kidney, mouse ascites tumor cells, wheat germ, and yeast, it suggests that the eIF2C gene is particularly important in eukaryotic tissues. It is widely expressed in reticular tissue and tumor tissue. Therefore, the abnormality of the translation initiation factor cofactor 28 gene and its expression product by the human of the present invention will cause various diseases, especially reticular tissue diseases and various tumors.
  • diseases include, but are not limited to: splenomegaly, hypersplenism, renal interstitial diseases such as interstitial nephritis, tubular acidosis, alcoholic cirrhosis, basal epithelial tumors, squamous epithelial tumors, mucinous tumors, fibers Tumors such as reticular histiocytoma, fibrohistiocytoma, malignant fibrohistiocytoma, lipoma, chondroma, hemangioma, lymphoma, hematopoietic tumors such as leukemia, neuroma, adenoma.
  • renal interstitial diseases such as interstitial nephritis, tubular acidosis, alcoholic cirrhosis, basal epithelial tumors, squamous epithelial tumors, mucinous tumors, fibers Tumors such as reticular histiocytoma, fibroh
  • These diseases include, but are not limited to: spina bifida, craniocerebral fissure, anencephaly malformation, cerebral bulge, foramencephalic malformation, congenital hydrocephalus, aqueduct malformation, dwarfism of cartilage hypoplasia, spinal epiphyseal dysplasia, false Cartilage hypoplasia, Langer-Giedion syndrome, funnel chest, gonad hypoplasia, congenital adrenal hyperplasia, upper urethra, cryptorchidism, with short stature Syndromes such as Conradi syndrome and Danbol t-Clos s syndrome, congenital glaucoma or cataract, congenital lens position abnormality, congenital blepharoplasia, retinal dysplasia, congenital optic nerve atrophy, congenital sensory neurological hearing loss, Cleft-hand and cracked feet, teratosis, Wi ll iams syndrome, Alag ille syndrome, Bayer syndrome.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) translation initiation factor cofactor 28.
  • Agonists enhance translation initiation factor cofactor 28 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or a membrane preparation expressing a translation initiation factor cofactor 28 can be cultured together with a labeled translation initiation factor cofactor 28 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of translation initiation factor cofactor 28 include selected antibodies, compounds, receptor deletions, and the like. Antagonists of translation initiation factor cofactor 28 can bind to translation initiation factor cofactor 28 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 exert its biology Features.
  • translation initiation factor cofactor 28 can be added to the bioanalytical assay to determine whether the compound is a compound by measuring the effect of the compound on the interaction between translation initiation factor cofactor 28 and its receptor. Antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to translation initiation factor cofactor 28 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, the 28 molecules of translation initiation cofactors should generally be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies directed against a translation initiation factor cofactor 28 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • polyclonal antibodies can be obtained by directly injecting immunized animals (such as rabbits, mice, rats, etc.) with translation initiation factor cofactor 28.
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's Agent.
  • Techniques for preparing monoclonal antibodies for translation initiation factor cofactor 28 include, but are not limited to, hybridoma technology (Kohler and Mistein. Nature, 1975, 256: 495-497), three Tumor technology, human B-cell hybridoma technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions and non-human-derived variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851).
  • the existing technology for producing single chain antibodies (US Pat No. 4946778) can also be used to produce single chain antibodies against translation initiation factor cofactor 28.
  • Antibodies against translation initiation factor cofactor 28 can be used in immunohistochemical techniques to detect translation initiation factor cofactor 28 in biopsy specimens.
  • Monoclonal antibodies that bind to translation initiation factor cofactor 28 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. Such as translation initiation factor cofactor 28.
  • High affinity 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 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 translation initiation factor cofactor 28 positive cell.
  • the antibodies of the present invention can be used to treat or prevent diseases related to translation initiation factor cofactor 28.
  • Administration of an appropriate dose of antibody can stimulate or block the production or activity of translation initiation factor cofactor 28.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of translation initiation factor cofactor 28.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of translation initiation factor cofactor 28 detected in the test can be used to explain the importance of translation initiation factor cofactor 28 in various diseases and to diagnose diseases in which translation initiation factor cofactor 28 plays a role.
  • 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 the translation initiation factor cofactor 28 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 non-expression or abnormal / inactive expression of translation initiation factor cofactor 28.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express a variant translation initiation factor cofactor 28 to inhibit endogenous translation initiation factor cofactor 28 activity.
  • a variant translation initiation cofactor 28 may be shortened, Co-factor 28, a translation initiation factor lacking the signaling domain, lacks signaling activity, although it can bind to downstream substrates.
  • the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of translation initiation factor cofactor 28.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding a translation initiation factor cofactor 28 into a cell.
  • Methods for constructing a recombinant viral vector carrying a polynucleotide encoding a translation initiation factor cofactor 28 can be found in existing literature (Sambrook, et al.).
  • a polynucleotide encoding the translation initiation factor cofactor 28 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 translation initiation factor cofactor 28 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 phosphoramidite chemical synthesis to synthesize oligonucleotides.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA.
  • This DNA sequence has been integrated downstream of the vector's RNA polymerase promoter.
  • it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • a polynucleotide encoding a translation initiation factor cofactor 28 can be used to diagnose diseases related to the translation initiation factor cofactor 28.
  • the polynucleotide encoding the translation initiation factor cofactor 28 can be used to detect the expression of the translation initiation factor cofactor 28 or the abnormal expression of the translation initiation factor cofactor 28 in a disease state.
  • the DNA sequence encoding the translation initiation factor cofactor 28 can be used to hybridize biopsy specimens to determine the expression of the translation initiation factor cofactor 28.
  • Hybridization techniques include Southern blotting, Northern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Co-factor with translation initiation factor The transcription of the transcription initiation factor cofactor 28 can also be detected by RNA-polymerase chain reaction (RT-PCR) in vitro amplification of specific primers.
  • Detecting mutations in the translation initiation factor cofactor 28 gene can also be used to diagnose translation initiation factor cofactor 28-related diseases.
  • Translation initiation factor cofactor 28 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type translation initiation factor cofactor 28 DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR, and in situ hybridization. In addition, mutations may affect the expression of proteins. Therefore, Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, the specific loci of each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) can be used to mark chromosome locations. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these D sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequences can be located 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.
  • 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, Mendelian Inherance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions. Next, the differences in 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.
  • the cDNA accurately mapped to the disease-related chromosomal region can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • 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.
  • the translation initiation factor cofactor 28 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dose range of translation initiation factor cofactor 28 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.
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • the determined cDNA sequence was compared with the existing public D-sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0581G05 was new DNA.
  • the inserted cDNA fragments contained in this clone were determined in both directions by synthesizing a series of primers.
  • the results showed that the 0581G05 clone contained a full-length cDNA of 2951bp (as shown in Seq ID NO: l), and a 759bp open reading frame (0RF) from 878bp to 1636bp, encoding a new protein (such as Seq ID NO : Shown in 2).
  • This clone pBS_0581G05 and the encoded protein was named translation initiation factor cofactor 28.
  • Example 2 Homologous search of cDNA clones
  • CDNA was synthesized using fetal brain total RNA as a template and ol igo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, the following primers were used for PCR amplification:
  • Pr imerl 5'- GATCAAAGCTACAGCAAGATCTGC-3 '(SEQ ID NO: 3)
  • Primer 2 5'- CACAGTTTTAGTTTAATAAAAGG -3 '(SEQ ID NO: 4)
  • Priraerl is a forward sequence starting at the lbp of the 5th end of SEQ ID NO: 1;
  • Primer2 is the 3 'reverse sequence in SEQ ID NO: 1,
  • Amplification conditions 50 mmol / L KC1, 10 mmol / L Tri s-Cl, (pH 8.5.5), 1.5 mmol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol in a reaction volume of 50 ⁇ 1 Primer, 1U Taq DNA Polymerization P CN00 / enzyme (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94. C 30sec; 55 ° C 30sec; 72 ° C 2min.
  • ⁇ -actin was set as a positive control and template blank was set as a negative control.
  • the amplified product was purified using a QIAGEN kit and ligated to a PCR vector (Invitrogen product) using a TA cloning kit.
  • the DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as the l-2951bp shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of the expression of the translation initiation factor cofactor 28 gene
  • the total RM nal was extracted by a one-step method. Biochem 1987, 162, 156-159]. This method involves acid guanidinium thiocyanate phenol-chloroform extraction.
  • the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ) And centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The obtained RM precipitate was washed with 70% ethanol, dried and dissolved in water.
  • RNA was synthesized by electrophoresis on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (PH7.0)-5 mM sodium acetate-ImM EDTA-2.2M formaldehyde. It was then transferred to a nitrocellulose membrane.
  • the DNA probe used was the PCR-amplified translation initiation factor cofactor 28 coding region sequence (878bp to 1636bp) shown in FIG.
  • Primer3 5'- CCCCATATGATGGATGGCCACCCCAGCCGGTAC-3 '(Seq ID No: 5)
  • Primer4 5'- CCCAAGCTTTCACCGAAAATACATCGTGTGCTG-3' (Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and Hindlll restriction sites, respectively.
  • the coding sequences of the 5 'and 3' ends of the gene of interest are followed, respectively.
  • the Ndel and Hindlll restriction sites correspond to the selectivity within the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Digestion site.
  • the PCR reaction was performed using the PBS-0581G05 plasmid containing the full-length target gene as a template.
  • PCR reaction conditions are: total volume 50 ⁇ 1
  • the pBS-0581G05 plasmid contained 10 pg, and the primers Primer-3 and Primer-4 were l Opmol and Advantage polymerase Mix (Clontech) 1 ⁇ 1, respectively.
  • Cycle parameters 94 ° C 20s, 60. C 30s, 68 ° C 2 min, 25 cycles in total.
  • Ndel and Hindl11 were used to double-digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligated product was transformed into E.
  • Polypeptide synthesizer (product of PE company) was used to synthesize the following polypeptides specific for translation initiation factor cofactor 28: NH 2 -Met-Asp-Gly-Hi s-Pro-Ser-Arg-Tyr-Cys-Ala-Thr- Va l-Arg-Va l-Gln-COOH (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 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. ⁇ Using a 15 g / ml bovine serum albumin peptide complex-coated titer plate 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 Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography. The immunoprecipitation method proved that the purified antibody could specifically bind to translation initiation factor cofactor 28.

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Abstract

L'invention concerne un nouveau polypeptide, un facteur auxiliaire 28 du facteur de démarrage de la traduction, 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 le facteur auxiliaire 28 du facteur de démarrage de la traduction.
PCT/CN2000/000382 1999-10-28 2000-10-27 Nouveau polypeptide, facteur auxiliaire 28 du facteur de demarrage de la traduction, et polynucleotide codant pour ce polypeptide Ceased WO2001031001A1 (fr)

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AU12646/01A AU1264601A (en) 1999-10-28 2000-10-27 A novel polypeptide, a translation initiation factor helper factor 28 and the polynucleotide encoding the polypeptide

Applications Claiming Priority (2)

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CN99119888A CN1302874A (zh) 1999-10-28 1999-10-28 一种新的多肽——翻译起始因子辅助因子28和编码这种多肽的多核苷酸
CN99119888.3 1999-10-28

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117877580A (zh) * 2023-12-29 2024-04-12 深药科技(苏州)有限公司 基于深度语言模型的多肽关键位点预测方法、设备和介质

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] 25 June 1998 (1998-06-25), Database accession no. AC005355 *
DATABASE GENBANK [online] 30 October 1998 (1998-10-30), Database accession no. AC002036 *
GENE, vol. 211, no. 2, 12 May 1998 (1998-05-12), pages 187 - 194 *
GENOMICS, vol. 60, no. 3, 15 September 1999 (1999-09-15), pages 295 - 308 *
GENOMICS, vol. 61, no. 2, 15 October 1999 (1999-10-15), pages 210 - 218 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117877580A (zh) * 2023-12-29 2024-04-12 深药科技(苏州)有限公司 基于深度语言模型的多肽关键位点预测方法、设备和介质

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