WO2002000822A2 - Nouveau polypeptide, sous-unite humaine 2 $g(b) 16.5 du facteur d'initiation necessaire a la synthese proteique, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, sous-unite humaine 2 $g(b) 16.5 du facteur d'initiation necessaire a la synthese proteique, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002000822A2
WO2002000822A2 PCT/CN2001/000818 CN0100818W WO0200822A2 WO 2002000822 A2 WO2002000822 A2 WO 2002000822A2 CN 0100818 W CN0100818 W CN 0100818W WO 0200822 A2 WO0200822 A2 WO 0200822A2
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polypeptide
polynucleotide
protein synthesis
subunit
initiation factor
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PCT/CN2001/000818
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Chinese (zh)
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WO2002000822A3 (fr
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Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc.
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Priority to AU87524/01A priority Critical patent/AU8752401A/en
Publication of WO2002000822A2 publication Critical patent/WO2002000822A2/fr
Publication of WO2002000822A3 publication Critical patent/WO2002000822A3/fr

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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

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide " ⁇ ⁇ protein synthesis initiation factor 2 P subunit 16. 5, and a polynucleotide sequence encoding the polypeptide. The present invention also relates to Preparation method and application of the polynucleotide and polypeptide. TECHNICAL BACKGROUND
  • the ternary complex binds directly to the 40S subunit, and this process does not require the presence of mRNA.
  • the 40S subunit-ternary complex binds to mRNA in the presence of eIF3.
  • one molecule of ATP is hydrolyzed to provide energy.
  • a complete biologically active protein translation initiation complex is formed, and the complex moves along the mRNA toward the start codon AUG to form 80S ribosomes.
  • 80S ribosome When the translationally active 80S ribosome is formed, eIF2 and eIF3 are released from the complex, and both the P and A positions on the ribosome are in the correct posture to promote the smooth progress of the protein synthesis process.
  • eIF2 protein synthesis initiation factor eIF2 plays a very important regulatory role in the formation of the initiation complex.
  • the eIF2 protein has been cloned from rabbits and humans, and its structure and biological function have been studied in detail.
  • eIF2 protein is a protein complex formed by five different subunits, namely alpha subunit, beta subunit, gamma subunit, delta subunit, and epsilon subunit. These five different subunits play different regulatory roles in the process of protein in vivo activity. The study found that eIF2 0 subunit may play a more important role in the interaction between protein and RNA.
  • the amino acid sequence of the protein contains a GTP binding site, a zinc finger protein domain and a highly variable N-terminal region.
  • the GTP binding site is the binding of protein to GTP, which promotes the mutual conversion of GTP and GDP into an important role site for protein synthesis to provide the energy required; while the zinc finger protein domain and the highly variable region at the N-terminus are the interaction between protein and RNA It plays an important regulatory role in the process. Mutation or abnormal expression of this protein subunit will lead to heterogeneous synthesis of protein synthesis initiation complex in vivo Often affect the synthesis of related proteins, and then cause various related developmental and metabolic disorders
  • the eIF2 P subunit has been cloned from rabbits and humans, and its structure and function have been analyzed.
  • Pathak et al. Cloned an eIF2 ⁇ subunit from humans and found that the protein subunit contains a GTP binding site, a zinc finger protein domain, and a highly variable N-terminal region. This protein has significant ATP and tRNA binding activities. It is involved in regulating the formation of protein synthesis initiation complexes in the body, and its mutation or abnormal expression will directly lead to abnormal synthesis of relevant tissue proteins in the body.
  • eIF2 plays an important regulatory role in the formation of the protein synthesis starting complex, and its constituent ⁇ subunit plays an important role in this process. Mutations or abnormal expression of this subunit will affect the protein's function in vivo, directly affect the formation of protein synthesis initiation complexes, and then affect the synthesis and function of related proteins in the body, thereby triggering various related development and The occurrence of metabolic disorders, tumors and cancers of related tissues and organs.
  • the human protein synthesis initiation factor 2 ⁇ subunit 16. 5 protein plays an important role in regulating important functions of the body such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, so the art has been More human protein synthesis initiation factor 2
  • New human protein synthesis initiation factor 2 ⁇ subunit 16. 5 The isolation of protein-coding genes also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for the disease, so it is important to isolate its coding for DM. Object of the invention
  • 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 human protein synthesis initiation factor 2 P subunit 16.5.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human protein synthesis initiation factor 2 P subunit 16.5.
  • Another object of the present invention is to provide a method for producing a human protein synthesis initiation factor subunit 16.5.
  • Another object of the present invention is to provide an antibody against the polypeptide-human protein synthesis initiation factor 2 ⁇ subunit 16.5 of the present invention.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the polypeptide-human protein synthesis initiation factor 2 ⁇ subunit 16.5 of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities in the human protein synthesis initiation factor subunit 16.5. Summary of invention
  • the present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 332-784 in SEQ ID NO: 1; and (b) a sequence having positions 1-972 in SEQ ID NO: 1 Sequence of bits.
  • the present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit human protein synthesis initiation factor 2 beta subunit 16.5 protein activity, which comprises utilizing the polypeptide of the invention.
  • This invention also It relates to compounds obtained by this method.
  • the present invention also relates to a method for in vitro detection of a disease or disease susceptibility related to abnormal expression of a human protein synthesis initiation factor 2 p subunit 16.5 protein, comprising detecting the polypeptide or a polynucleotide sequence encoding the same in a biological sample Mutations, or the amount or biological activity of a polypeptide of the invention in a biological sample.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to polypeptides and / or polynucleotides of the present invention prepared for the treatment of cancer, developmental disease or immune disease or other diseases caused by abnormal expression of human protein synthesis initiation factor 2 ⁇ subunit 16.5. Use of medicine.
  • FIG. 1 is a comparison diagram of gene chip expression profiles of human protein synthesis initiation factor 2 ⁇ subunit 16.5 and human protein synthesis initiation factor 2 ⁇ subunit.
  • the upper graph is a graph of the expression profile of the human protein synthesis initiation factor 2 ⁇ subunit 16. 5 and the lower graph is the graph of the expression profile of the human protein synthesis initiation factor 2 ⁇ subunit.
  • 1-bladder mucosa 2-PMA + Ecv304 cell line, 3-LPS + Ecv304 cell line thymus, 4-normal fibroblasts 1024NC.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated human protein synthesis initiation factor 2 ⁇ subunit 16.5. 16kDa is the molecular weight of the protein. The arrow indicates the isolated protein band.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DM or RM, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule
  • polypeptide or “protein” is not meant to limit the amino acid sequence to the complete natural amino acid associated with the protein molecule.
  • a “variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion means that an alteration in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that, when combined with human protein synthesis initiation factor 2 beta subunit 16.5, can cause the protein to change, thereby regulating the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to human protein synthesis initiation factor 2 beta subunit 16.5.
  • Antagonist refers to an organism that can block or regulate human protein synthesis initiation factor 2 ⁇ subunit 16.5 when combined with human protein synthesis initiation factor 2 ⁇ subunit 16.5.
  • Molecularly active or immunologically active molecule may include proteins, nucleic acids, carbohydrates or any other molecule that can bind to human protein synthesis initiation factor 2 beta subunit 16.5.
  • Regular refers to a change in the function of the human protein synthesis initiation factor 2 ⁇ subunit 16.5, including an increase or decrease in protein activity, a change in binding characteristics, and a human protein synthesis initiation factor 2 ⁇ subunit 16.5. Of any other biological, functional or immune properties.
  • Substantially pure means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify the human protein synthesis initiation factor 2 ⁇ subunit 16.5 using standard protein purification techniques.
  • Substantially pure human protein synthesis initiation factor 2 ⁇ subunit 16.5 can produce a single main band on a non-reducing polyacrylamide gel.
  • Human protein synthesis initiation factor 2 ⁇ subunit 16. 5 Purity of the polypeptide The amino acid sequence can be analyzed.
  • Complementary refers to a polynucleotide that naturally binds by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence "C-T-G- ⁇ ” may be combined with the complementary sequence "G-ACT”.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern imprinting or Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences according to different methods, such as the Clus ter method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). 0 The Clus ter method groups each group by checking the distance between all pairs. The sequences are arranged in clusters. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula:
  • the percent identity between nucleic acid sequences can also be determined by the Clus ter method or by methods known in the art such as Jotun Hein (Hein L, (1990) Methods in enzymology 183: 625-645).
  • Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitution for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DM or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to HFP or a chemical modification of its nucleic acid. This chemical modification may be a substitution of a hydrogen atom with a fluorenyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ) ') 2 and?, Which can specifically bind to the human protein synthesis initiation factor 2 ⁇ subunit 16.5 epitope.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not part of its natural environment, they are still isolated.
  • 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 human protein synthesis initiation factor 2 ⁇ subunit 16. means human protein synthesis initiation factor 2 ⁇ subunit 16. 5 is substantially free of other proteins, lipids, and sugars naturally associated with it. Or other substances. Those skilled in the art can purify human protein synthesis initiation factor 2 ⁇ subunit 16.5 using standard protein purification techniques. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. Human protein synthesis initiation factor 2 ⁇ subunit 16.5 The purity of the polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human protein synthesis initiation factor 2 ⁇ subunit 16. 5, 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 can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques.
  • polypeptide of the invention may be glycosylated, or it may be non-glycosylated.
  • the polypeptides of the invention may also include or exclude the initial methionine residue.
  • the invention also includes fragments, derivatives and analogs of the human protein synthesis initiation factor 2 ⁇ subunit 16.5.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human protein synthesis initiation factor 2 ⁇ subunit 16.5 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 the genetic codon; or ( ⁇ ) such a type in which a group on one or more amino acid residues is replaced by another group Or (III) such that the mature polypeptide is fused with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) such that the additional amino acid sequence is fused into the mature A polypeptide sequence (such as a leader sequence or a secreted sequence or a sequence used to purify the polypeptide or a protein sequence) formed by the polypeptide.
  • 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 CDM library of human fetal brain tissue. It contains a polynucleotide sequence of 972 bases in length and its open reading frames 332-784 encode 150 amino acids.
  • this peptide has a similar expression profile to the human protein synthesis initiation factor 2 ⁇ subunit, and it can be inferred that the human protein synthesis initiation factor 2 ⁇ subunit 16.5 has human protein synthesis initiation Factor 2 beta subunits have similar functions.
  • the polynucleotide of the present invention may be in the form of DNA or RM.
  • DNA forms include cDNA, 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 invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions.
  • “strict conditions” means: (1) in the lower Hybridization and elution at ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) adding a denaturant such as 50% (v / v) formamide during hybridization, 0.1% calf serum / 0.1% Fico ll, 42 ° C, etc .; or (3) hybridization occurs only when the identity between the two sequences is at least 95%, and more preferably 97% .
  • 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 in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding the human protein synthesis initiation factor 2 ⁇ subunit 16.5.
  • 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 human protein synthesis initiation factor 2 ⁇ subunit 16.5 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 cloned polynucleosides with common structural characteristics Acid fragments.
  • 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 DM 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 raRM from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • Various methods have been used to extract mRNA, and kits are also commercially available (Qiagene).
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manua, Cold Sprue 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.
  • 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 a marker gene function; (3) determination of human protein synthesis initiation factor 2 ⁇ subunit 16.5 transcription (4) Detecting protein products expressed by genes through immunological techniques or measuring biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is any part of the polynucleotide of the present invention Homologous, at least 10 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, most 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 human protein synthesis initiation factor 2 ⁇ subunit 16.5 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). Wait.
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). Wait.
  • a method (Saiki, et al. Science 1985; 230: 1350-1354) using DNA technology to amplify DNA / RM is preferably used to obtain the gene of the present 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 / 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 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 human protein to synthesize an initiation factor 2 ⁇ subunit 16.5 coding sequence, and the recombinant technology to produce the present invention.
  • a method of inventing the polypeptide is not limited 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 human protein to synthesize an initiation factor 2 ⁇ subunit 16.5 coding sequence, and the recombinant technology to produce the present invention.
  • a polynucleotide sequence encoding a human protein synthesis initiation factor 2 ⁇ subunit 16.5 can 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 recombinant expression vectors.
  • 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 well known to those skilled in the art can be used to construct a gene containing human protein synthesis initiation factor 2 ⁇ .
  • Expression sequence for the subunit 16.5 D sequence and appropriate transcriptional / translational regulatory elements include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manua, Cold Spring Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • 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 DNA expression, usually about 10 to 300 base pairs, which 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 human protein synthesis initiation factor 2 P subunit 16.5 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a polynucleotide containing the polynucleotide or the recombinant vector.
  • Genetically engineered host cells 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. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence according to the present invention or a recombinant vector containing the DM 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 for expression or production Recombinant human protein synthesis initiation factor 2 beta subunit 16. 5 (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 initiation of protein synthesis is the most important step in protein synthesis in an organism.
  • the synthesis of protein chains requires the existence of multiple protein synthesis initiation factors. These different initiation factors are combined with some related energy substances and MA and other substances in the body to form corresponding protein synthesis initiation complexes.
  • the protein synthesis initiation factor eIF2 plays a very important regulatory role in the formation of the initiation complex.
  • the eIF2 P subunit may play a more important role in the role of proteins and RNA. Mutations or abnormal expression of this protein subunit will lead to abnormal synthesis of protein synthesis initiation complexes in the body, and then affect related proteins. Synthesis, which in turn triggers various related developmental and metabolic disorders. '
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of the human protein synthesis initiation factor 2 P subunit, and both have similar biological functions.
  • the polypeptide of the present invention is most important for the initiation of protein biosynthesis in cells, and it is combined with some related energy substances in the body and MA and other substances to form The corresponding protein synthesis starting complex is formed, and the normal initiation of genetic material in the mediator is translated into the corresponding protein product. Its abnormal expression usually causes various developmental and metabolic disorders, and produces related diseases.
  • the abnormal expression of the human protein synthesis initiation factor 2 ⁇ subunit 16. 5 of the present invention will produce various diseases, especially embryonic developmental disorders, growth disorders, tumors, inflammation, and immune diseases. Illnesses include, but are not limited to:
  • Fetal developmental disorders congenital abortion, cleft palate, limb loss, limb differentiation disorder, atrial septal defect, neural tube defect, congenital hydrocephalus, congenital glaucoma or cataract, congenital deafness
  • Growth and development disorders mental retardation, brain development disorders, skin, fat and muscular dysplasia, bone and joint dysplasia, various metabolic deficiencies, stunting, dwarfism, Cushing syndrome, Sexual retardation
  • Tumors gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, astrocytoma, ependymal tumor, glioblastoma, neurofibromas, colon cancer, bladder cancer , Endometrial cancer, fibroma, fibrosarcoma
  • Inflammation chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, cerebrospinal multiple sclerosis, glomerulonephritis, myocarditis, cardiomyopathy, atherosclerosis, gastric ulcer, cervicitis, Various infectious inflammations
  • Immune diseases Systemic lupus erythematosus, rheumatoid arthritis, bronchial asthma, urticaria, specific dermatitis, post-infection myocarditis, scleroderma, myasthenia gravis, Guillain-Barre syndrome, common variable immunodeficiency disease , Primary B-lymphocyte immunodeficiency disease, Acquired immunodeficiency syndrome
  • the abnormal expression of the human protein synthesis initiation factor subunit 16.5 of the present invention will also produce certain hereditary, hematological diseases and the like.
  • 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 various diseases, especially embryonic developmental disorders, growth disorders, tumors, inflammation, and immune diseases. , Some hereditary, hematological diseases, etc.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human protein synthesis initiation factor 2 beta subunit 16.5.
  • Agonists increase human protein synthesis initiation factor 2 ⁇ subunits 16. 5 Stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • a mammalian cell or a membrane preparation expressing human protein synthesis initiation factor 2 ⁇ subunit 16.5 can be cultured with a labeled human protein synthesis initiation factor 2 ⁇ subunit 16.5 in the presence of a drug. . The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human protein synthesis initiation factor 2 ⁇ subunit 16. 5 include selected antibodies, compounds Substances, receptor deletions, and the like. Antagonists of human protein synthesis initiation factor 2 (3 subunit 16.5 can bind to human protein synthesis initiation factor 2 p subunit 16.5 and eliminate its function, or inhibit the production of the polypeptide, or with the The active site binding of a polypeptide prevents the polypeptide from performing its biological function.
  • human protein synthesis initiation factor 2 ⁇ subunit 16.5 can be added to the bioanalytical assay, and the human protein synthesis initiation factor 2 ⁇ subunit 16.5 and its receptors can be determined by determining the compounds. The effect of this interaction is used to determine whether the compound is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Peptide molecules capable of binding to human protein synthesis initiation factor 2 ⁇ subunit 16.5 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, generally 16.5 molecules of human protein synthesis initiation factor 2 ⁇ subunits should 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 against the human protein synthesis initiation factor 2 beta subunit 16.5 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 injecting human protein synthesis initiation factor 2 ⁇ subunit 16.5 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including But it is not limited to Freund's adjuvant.
  • Techniques for preparing monoclonal antibodies to human protein synthesis initiation factor 2 P subunit 16. 5 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple 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 Pat No. 4946778) can also be used to produce single chain antibodies against human protein synthesis initiation factor 2 ⁇ subunit 16.5.
  • Antibodies against human protein synthesis initiation factor 2 ⁇ subunit 16. 5 can be used in immunohistochemical techniques to detect human protein synthesis initiation factor 2 ⁇ subunit 16.5 in biopsy specimens.
  • Monoclonal antibodies that bind to the human protein synthesis initiation factor 2 ⁇ subunit 16.5 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.
  • 5 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 sulfhydryl 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 In killing human protein synthesis initiation factor 2 ⁇ subunit 16. 5 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to the human protein synthesis initiation factor 2 P subunit 16.5.
  • Administration of appropriate doses of antibodies can stimulate or block the production or activity of human protein synthesis initiation factor 2 beta subunit 16.5.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human protein synthesis initiation factor 2 beta subunit 16.5. These tests are well known in the art and include FISH and radioimmunoassays.
  • the level of human protein synthesis initiation factor 2 ⁇ subunit 16.5 detected in the test can be used to explain the importance of human protein synthesis initiation factor 2 P subunit 16.5 in various diseases and for diagnosis of humans Diseases where protein synthesis initiation factor 2 ⁇ subunit 16.5 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.
  • Polynucleotides encoding the human protein synthesis initiation factor 2 beta subunit 16.5 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 human protein synthesis initiation factor 2 ⁇ subunit 16.5.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human protein synthesis initiation factor 2 ⁇ subunit 16.5 to inhibit endogenous human protein synthesis initiation factor 2 ⁇ subunit 16.5 activity.
  • a mutated human protein synthesis initiation factor 2 ⁇ subunit 16.5 may be a shortened human protein synthesis initiation factor 2 ⁇ subunit 16.5, which lacks a signaling functional domain, although it can interact with downstream substrates. Binding, but lacks signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of human protein synthesis initiation factor 2 ⁇ subunit 16.5.
  • Expression vectors derived from viruses such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding a human protein synthesis initiation factor 2 ⁇ subunit 16.5 into a cell .
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a human protein synthesis initiation factor 2 ⁇ subunit 16.5 can be found in the existing literature (Sambrook, et al.).
  • recombinant polynucleotides encoding human protein synthesis initiation factor 2 ⁇ subunit 16.5 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 human protein synthesis initiation factor 2 beta subunit 16.5 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RM molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for nucleic acid. Inward action.
  • Antisense RNA, DNA, and ribozymes can be obtained using any existing RM or DNA synthesis technology, such as the technology for the synthesis of oligonucleotides by solid-phase phosphoramidite chemical synthesis has been widely used.
  • 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 vector's RNA polymerase promoter. In order to increase the stability of a nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside linkages should use phosphate thioester or peptide bonds instead of phosphodiester bonds. ,
  • the polynucleotide encoding the human protein synthesis initiation factor 2 ⁇ subunit 16.5 can be used for the diagnosis of diseases related to the human protein synthesis initiation factor 2 ⁇ subunit 16.5.
  • a polynucleotide encoding a human protein synthesis initiation factor 2 ⁇ subunit 16.5 can be used to detect the expression of human protein synthesis initiation factor 2 ⁇ subunit 16.5 or the human protein synthesis initiation factor 2 ⁇ subunit in a disease state 16. 5 abnormal expression.
  • a DNA sequence encoding human protein synthesis initiation factor 2 ⁇ subunit 16.5 can be used to hybridize biopsy specimens to determine the expression of human protein synthesis initiation factor 2 ⁇ subunit 16.5.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • a part or all of the polynucleotides of the present invention can be used as probes to be fixed on a micro array or a DM chip (also known as a "gene chip") for analyzing differential expression analysis of genes and genetic diagnosis in tissues.
  • Human protein synthesis initiation factor 2 ⁇ subunit 16.5 specific primers for RM-polymerase chain reaction (RT-PCR) in vitro amplification can also detect the human protein synthesis initiation factor 2 ⁇ subunit 16.5 transcription products.
  • Human protein synthesis initiation factor 2 ⁇ subunit 16.5 mutant forms include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type human protein synthesis initiation factor 2 ⁇ subunit 16.5 DNA sequences Wait. 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. 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, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequences can be mapped on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells that contain the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • 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 CDM libraries.
  • Fluorescent in situ hybridization of cDM clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDM sequence-based PCR. Based on the resolution capabilities of current physical mapping and gene mapping technology, the CDM that is accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and 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 human protein synthesis initiation factor 2 ⁇ subunit 16.5 is administered in an amount effective to treat and / or prevent a specific indication. Human protein synthesis initiation factor 2 ⁇
  • the amount and range of subunits 16.5 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. Examples
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using Quik mRNA Isolat ion Kit (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA.
  • a Smart cDNA cloning kit (purchased from Clontech) was used to insert the cDNA fragment into the multicloning site of pBSK (+) vector (Clontech) to transform DH5a. The bacteria formed a CDM library.
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • cDNA sequence of one of the clones 0725F08 was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • CDNA was synthesized using fetal brain cell 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:
  • Primerl 5'- GGATGACGCACCCATGGCGCCTGG -3 '(SEQ ID NO: 3)
  • Primer2 5'- GTGTTTTTTGCTTTATTTATTCAG -3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Primer 2 is the 3, terminal reverse sequence of SEQ ID NO: 1.
  • Amplification conditions 50 mmol / L KCl, 1 (kmol / L Tris-HCl pH 8. 5, 1.5ramol / L MgCl 2 , 20 ( ⁇ mol / L dNTP, 1 Opmol primer, 1U Taq DNA polymerase (Clontech)).
  • a PE9600 DNA thermal cycler Perkin-Elmer 25 cycles according to the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2rain 0 During RT-PCR, ⁇ -act in was used as a positive control and template blank was used as a negative control.
  • QIAGEN was used as the amplification product.
  • the kit was purified and ligated to a PCR vector (Invitrogen product) using a TA cloning kit. DM sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as 1-972bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human protein synthesis initiation factor 2 ⁇ subunit 16.5 gene expression
  • RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] 0
  • This method involves acid guanidinium thiocyanate-chloroform extraction. That is, the tissue was homogenized with 4M guanidine isothiocyanate-25raM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1), centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • a 32P-labeled probe (approximately 2 x 10 6 cpm / ml) and a nitrocellulose membrane to which MA has been transferred are placed in a solution in Hybridize overnight at 42 ° C.
  • This solution contains 50% formamide-25raM KH 2 P0 4 (pH7. 4) -5 33 (-5 ⁇ 1) 61111 & £ 11 ;, 3 solutions and 20 ( ⁇ ⁇ / 1111 salmon sperm 0
  • the filter was washed in 1 X SSC-0. 1% SDS at 55 ° C. for 30 min. Then, it was analyzed and quantified by Phosphor Imager.
  • Example 4 Recombinant human protein synthesis initiation factor 2 ⁇ subunit 16.5 In vitro expression, isolation and purification
  • Pr imer 3 5'-CATGCTAGCATGGCTGCCTCCCAGTGTCTCTGC-3 '(Seq ID No: 5)
  • Pr imer4 5' -CCCGAATTCTTAAGGCTTTGAATAGCTATTCAT- 3 '(Seq ID No: 6)
  • the 5 ends of these two primers contain Nhel and EcoRI digestion respectively Sites, followed by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively.
  • the Nhel and EcoRI restriction sites correspond to the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865. 3) Selective endonuclease site.
  • the pBS-0725F08 plasmid of the target gene was used as a template for PCR reaction.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1, 10 PBS of PBS-0725F08 plasmid, Prima-3 and Primer-4 points, and 1 ⁇ l Optnol, Advantage polymerase Mix (Clontech) 1 ⁇ 1. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles. Nhel and EcoRI 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 coliform bacteria DH5 CC using the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 30 ⁇ ⁇ 1), positive clones were screened by colony PCR and sequenced. A positive clone (PET-0725F08) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method.
  • a peptide synthesizer (product of PE company) was used to synthesize the following human protein synthesis initiation factor 2 ⁇ 'subunit 16. 5 specific peptides:
  • polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin For methods, see: Avrameas, et al. Isa nochemi s try, 1969; 6:43. Rabbits were immunized with 4 mg of the hemocyanin-polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin-polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once.
  • a titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum.
  • Protein A-Sepharose was used to isolate total IgG from antibody-positive rabbit sera.
  • the peptide was bound to a cyanogen bromide-activated Sepharos B 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 the human protein synthesis initiation factor 2 ⁇ subunit 16.5.
  • Example 6 Application of the polynucleotide fragment of the present invention as a hybridization probe
  • the probes can be used to hybridize to the genome or CDM library of normal tissue or pathological tissue from different sources In order to identify whether it contains a polynucleotide sequence of the present invention and detect a homologous polynucleotide sequence, the probe may further be used to detect the polynucleotide sequence of the present invention or a homologous polynucleotide sequence thereof in normal tissue or Whether the expression in pathological tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by using a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern imprinting, Northern blotting, and copying methods. They all use the same steps to immobilize the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature), so that the hybridization background is reduced and only strong specific signals are retained.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, then the primary probe should not be used;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (lock):
  • Probe 2 which belongs to the second type of probe, is equivalent to the replacement mutant sequence of the gene fragment of SEQ ID NO: 1 or its complementary fragment (41Nt):
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membrane nitrocellulose membrane
  • the sample membrane was placed in a plastic bag, and 3-lOrag pre-hybridization solution (lOxDenhardt-s; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA)) was added. After sealing the mouth of the bag, shake at 68 ° C for 2 hours.
  • 3-lOrag pre-hybridization solution lOxDenhardt-s; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA)
  • Gene microarrays or DNA microarrays are new technologies currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; finding and screening for tissue specificity New genes, especially those related to diseases such as tumors; Diagnosis of diseases, such as hereditary diseases. The specific method steps have been reported in the literature.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were respectively amplified by PCR, and the concentration of the amplified product was adjusted to about 500ng / ul after purification.
  • the spots were spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 ⁇ m.
  • the spotted slides were hydrated and dried, cross-linked in a UV cross-linker, and dried after elution to fix the DNA on the glass slides to prepare chips.
  • the specific method steps have been reported in the literature.
  • the sample post-processing steps in this embodiment are:
  • the above specific tissues are bladder mucosa, PMA + Ecv304 cell line, LPS + Ecv304 cell line thymus, normal fibroblasts 1024NC, Fibroblast, growth factor stimulation, 1024NT, scar-like fc growth factor stimulation 1013HT, scar into fc not stimulated with growth factors, 1013HC, bladder cancer cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunal adenocarcinoma, cardia cancer. Based on these 17 Cy3 / Cy5 ratios, a histogram is drawn (Figure 1). It can be seen from the figure that the expression profiles of the human protein synthesis initiation factor 2 ⁇ subunit 16.5 and the human protein synthesis initiation factor 2 ⁇ subunit according to the present invention are very similar.

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Abstract

L'invention concerne un nouveau polypeptide, une sous-unité humaine 2 β 16.5 du facteur d'initiation nécessaire à la synthèse protéique, et un polynucléotide codant 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 troubles du développement, 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 la sous-unité humaine 2 β 16.5 du facteur d'initiation nécessaire à la synthèse protéique.
PCT/CN2001/000818 2000-05-24 2001-05-21 Nouveau polypeptide, sous-unite humaine 2 $g(b) 16.5 du facteur d'initiation necessaire a la synthese proteique, et polynucleotide codant ce polypeptide WO2002000822A2 (fr)

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CN 00115818 CN1324845A (zh) 2000-05-24 2000-05-24 一种新的多肽——人蛋白合成起始因子2β亚单位16.5和编码这种多肽的多核苷酸

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EP2033953A1 (fr) 2002-02-15 2009-03-11 Glaxo Group Limited Modulateurs des récepteurs vanilloides
JP2008047880A (ja) * 2006-08-11 2008-02-28 Samsung Electronics Co Ltd 半導体素子のヒューズボックス及びその形成方法
US9662327B2 (en) 2011-06-17 2017-05-30 Agios Pharmaceuticals, Inc Phenyl and pyridinyl substituted piperidines and piperazines as inhibitors of IDH1 mutants and their use in treating cancer

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