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WO2001038367A1 - Nouveau polypeptide - proteine 66 humaine associee a ras, et polynucleotide codant pour ledit polypeptide - Google Patents

Nouveau polypeptide - proteine 66 humaine associee a ras, et polynucleotide codant pour ledit polypeptide Download PDF

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Publication number
WO2001038367A1
WO2001038367A1 PCT/CN2000/000463 CN0000463W WO0138367A1 WO 2001038367 A1 WO2001038367 A1 WO 2001038367A1 CN 0000463 W CN0000463 W CN 0000463W WO 0138367 A1 WO0138367 A1 WO 0138367A1
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Prior art keywords
polypeptide
polynucleotide
binding protein
human ras
sequence
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PCT/CN2000/000463
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Bioroad Gene Development Ltd. Shanghai
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Application filed by Bioroad Gene Development Ltd. Shanghai filed Critical Bioroad Gene Development Ltd. Shanghai
Priority to AU15109/01A priority Critical patent/AU1510901A/en
Publication of WO2001038367A1 publication Critical patent/WO2001038367A1/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
    • 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, a human Ras binding protein 66, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and the polypeptide. Background technique
  • growth factors After some growth factors bind to their corresponding receptors, they activate the receptor's tyrosine protein kinase, which rephosphorylates the tyrosine residues of the target protein, and then affects gene expression through a series of phosphorylation cascades. .
  • the characteristic of this pathway is that it does not need to pass the G protein, but completes the transmembrane signal transmission through the activation of the receptor's own tyrosine protein kinase.
  • growth factor receptors Eight types have been found to belong to this signaling pathway: epidermal growth factor receptor, platelet growth factor receptor, hepatocyte growth factor receptor, nerve growth factor receptor, vascular endothelial growth factor receptor, insulin Growth factor receptor, insulin receptor, macrophage colony-stimulating factor receptor.
  • the Ras protein is a product of the ras gene, consisting of 190 amino acids and a molecular weight of 21KD. Ras is covalently bonded to isopentenyl to anchor it on the cytoplasmic side of the plasma membrane. Ras protein is not a protein kinase. Ras activates Ras when it is combined with GTP, and inactivates Ras when it is combined with GDP, so the function of Ras can be regarded as a molecular switch. When Ras is activated, the Raf protein located in the cytoplasm tends to the inside of the plasma membrane cell and binds to Ras. Ras activates Raf.
  • Raf is a serine / threonine protein kinase that phosphorylates serine / threonine residues on proteins.
  • the process of transmitting downstream signals after Raf activation involves many phospho- / threonine residue phosphorylation cascades.
  • One of the most important is mitogen-activated protein (MAP) kinase (MAPK).
  • MAPK mitogen-activated protein kinase
  • Activated MAPK enters the nucleus and regulates the transcription and expression of some genes, such as jun and Elk-1 or some protein kinases and other proteins.
  • Raf protein has three highly conserved regions (CR1, CR2, CR3), where the CR1 structure has a Ras-binding domain (RBD).
  • Ras-binding domains Ras-binding domains
  • Studies on the structure of Ras binding domain (RBD) have shown that its secondary structure is similar to ubiquitin superfolded crusts.
  • Related literature Biochem Biophys Res Commun 1997 Sep 18; 238 (2): 425-9); (Proc Natl Acad Sci USA 1996 Feb 20; 93 (4): 1577-81); (J Biol Chem 1995 Feb 17; 270 (7): 2901-5)
  • Ras proteins From yeast to mammals, Ras proteins have been shown to regulate the actin cytoskeleton. Members of the Ras protein family have leucine-rich repeat domains (LRRs), and gelsolin-like domains have been shown to be involved in Ras and actin binding. (Biochem Biophys Res Commun 1999 Apr 2; 257 (1): 111-6)
  • Ras proteins are a class of binding proteins contain R as binding domains (Ras- binding domain, RBD), which is leucine-rich repeat domain (LRcccccccccccccccccccccccccccccRs) structure, has been shown to bind to Ras proteins play a regulatory signals The important role of conduction.
  • RBD binding domains
  • sur-8 that encodes a Ras binding protein. Decreased sur-8 function results in suppression of mutated Ras gene expression and significantly increases the phenotype of mpk-1 MAP kinase and ksr-1 mutations.
  • the sur-8 gene may be downstream or parallel to ras, but it must be upstream of raf.
  • the sur-8 gene encodes a protein rich in leucine repeats.
  • the SUR-8 protein has a direct interaction with Ras, which is a Ras-binding protein. (Cell. 1998 Jul 10; 94 (1): 119-30)
  • Ras protein is a member of the GTP binding protein superfamily. It has a conserved GTP binding motif. GTP binding motif has a wide range of biological functions. It regulates cell proliferation, controls signal transduction, and various proteins in the body Expression in vivo. The abnormal expression of these proteins will lead to abnormal proliferation of tissue cells and abnormal expression of proteins, which will cause various related diseases, such as: various malignant tumors and cancers, various developmental disorders, and various immune system diseases.
  • Ras binding protein can play an important role in the body by regulating the expression level of Ras protein, including the abnormal proliferation of tissue cells and the abnormal expression process of the protein, so that it can be used to control cell growth, division, death, and differentiation It forms tissues and various life processes, and can be further used for the diagnosis and treatment of various related diseases, such as: various malignant tumors and cancers, various development disorders, various immune system diseases, etc.
  • the polypeptide of the present invention was inferred and identified as a new human Ras-binding protein 66 (HRasBP66).
  • the homologous protein was human Ras-binding protein SUR-8, and the protein number was AF068920.
  • the human Ras-binding protein 66 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 there has been a need in the art to identify more involved in these processes.
  • Human Ras-binding protein 66 protein particularly the amino acid sequence of this protein.
  • the isolation of the new Ras-binding protein 66 protein-encoding gene has also been confirmed for the study. It provides a basis for determining 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 diseases, so it is important to isolate its coding DNA. Disclosure of 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 Ras binding protein 66.
  • Another object of the present invention is to provide a genetically engineered host cell comprising a polynucleotide encoding a human Ras binding protein 66.
  • Another object of the present invention is to provide a method for producing human Ras-binding protein 66.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention-human Ras binding protein 66.
  • Another object of the present invention is to provide analog compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention-human Ras binding protein 66.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of human Ras binding protein 66.
  • the present invention relates to an isolated polypeptide, which is of human origin, and includes: a polypeptide having the amino acid sequence of SEQ ID D. 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 60-1868 in SEQ ID NO: 1; and (b) a sequence having 1-2840 in SEQ ID NO: 1 Sequence of bits.
  • the invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said Host cell and method of preparing the polypeptide of the invention by recovering the expression product.
  • a vector in particular an expression vector, containing the polynucleotide of the invention
  • a host cell genetically engineered with the vector including a transformed, transduced or transfected host cell
  • a method comprising culturing said Host cell and method of preparing the polypeptide of the 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 the activity of human Ras binding protein 66 protein, which comprises utilizing the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for detecting a disease or susceptibility to disease associated with abnormal expression of human Ras binding protein 66 protein in vitro, which comprises detecting a mutation in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, or The amount or biological activity of a polypeptide of the invention in a sample.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the polypeptide of the present invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of human Ras binding protein 66.
  • Nucleic acid sequence refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and can also refer to genomic or synthetic DNA or RNA, which can be single-stranded or double-stranded, representing the sense strand or Antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • 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” or “addition” refers to changes in the amino acid sequence or nucleotide sequence that result in The molecule is increased compared to one or more amino acids or nucleotides. “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 in appropriate animals or cells and to bind to specific antibodies.
  • An "agonist” refers to a molecule that, when bound to human Ras-binding protein 66, causes 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 human Ras binding protein 66.
  • Antagonist refers to a molecule that can block or regulate the biological or immunological activity of human Ras-binding protein 66 when bound to human Ras-binding protein 66.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind human Ras binding protein 66.
  • Regular refers to a change in the function of human Ras-binding protein 66, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological, functional, or immunological changes in human Ras-binding protein 66.
  • Substantially pure ' means essentially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify human Ras binding protein 66 using standard protein purification techniques. Essentially pure The human Ras binding protein 66 can generate a single main band on a non-reducing polyacrylamide gel. The purity of the human Ras binding protein 66 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence C-T-G-A
  • complementary sequence G-A-C-T
  • 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. The inhibition of such 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 conditions with reduced stringency allow non-specific binding, because conditions with reduced stringency require that the two sequences bind to each other as either specific or selective interactions.
  • 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 percentage identity can be determined electronically, such as by the MEGALIGN program (Lasergene software package, DNASTAR, Inc., Madison Wis.). MEGALIGN The program can compare two or more sequences according to different methods such as the Cluster method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244).
  • the Clus ter method arranges groups of sequences into clusters by checking the distance between all pairs. 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 number of matching residues between sequence A and sequence B
  • the number of residues in sequence A-the number of spacer residues in sequence A-the number of spacer residues in sequence B can also be determined by the Cluster method or by methods known in the art such as Jotun Hein. ⁇ T., (1990) Methods in erazumology 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 substitutions 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 DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, 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? ⁇ It can specifically bind to the epitope of human Ras-binding protein 66.
  • 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 occurs naturally).
  • a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, 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 a component 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 existing in the natural state. .
  • isolated human Ras-binding protein 66 means that human Ras-binding protein 66 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify human Ras binding protein 66 using standard protein purification techniques. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the human Ras-binding protein 66 peptide can be analyzed by amino acid sequences.
  • the present invention provides a new polypeptide, human Ras binding protein 66, 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. 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 human Ras binding protein 66.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the human Ras binding protein 66 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 (II) a type in which a group on one or more amino acid residues is substituted 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 full-length polynucleotide sequence of 2840 bases, and its open reading frame (60-1868) encodes 602 amino acids. According to the amino acid sequence homology comparison, it was found that this polypeptide has the human Ra s binding protein SUR-8. 70% homology, it can be deduced that the human Ras binding protein 66 has a similar structure and function of the human Ras binding protein SUR-8.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 in the present invention, but which differs from the coding region sequence shown in SEQ ID NO: 1.
  • 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) added during hybridization Use a denaturant, such as 50% (v / v) formamide, 0.1% calf serum / 0.1Wicoli, 42 ° C, etc .; or (3) the identity between the two sequences is at least 95%, More preferably, hybridization does not occur until 97% or more.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 cores. 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 human Ras binding protein 66.
  • 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 Ras-binding protein 66 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 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 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 raRNA 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., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include, but are not limited to: (DDNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of the level of human Ras-binding protein 66 transcripts; (4) immunity Technology or measuring biological activity to detect protein products expressed by genes. The above methods can be used alone 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 DM sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product expressed by the human Ras-binding protein 66 gene.
  • a method of applying a PCR technique to amplify DNA / RNA 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 sequences of the gene of the present invention obtained as described above, or various DNA fragments can be used It is determined 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 must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell genetically engineered using the vector of the present invention or directly using a human Ras binding protein 66 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology.
  • a polynucleotide sequence encoding human Ras-binding protein 66 may be inserted into a vector to form a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain origins of replication, promoters, marker genes, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human Ras-binding protein 66 and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation, a transcription terminator, and the like. 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 replication initiation point, polyoma enhancers and adenoviral enhancers on the late side of the replication initiation point.
  • 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 for eukaryotic cell culture. And green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance for eukaryotic cell culture.
  • GFP green fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human Ras-binding protein 66 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 ( 12 method, the steps used are well known in the art.
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and lipid Plastid packaging, etc.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human Ras-binding protein 66 (Scence, 1984; 224: 1431). Generally, the following steps are taken:
  • 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.
  • recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. 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 chromat
  • FIG. 1 is a comparison diagram of amino acid sequence homology between the Ras-binding protein 66 and the human Ras-binding protein SUR-8 of the present invention.
  • the upper sequence is human Ras-binding protein 66
  • the lower sequence is human Ras-binding protein SUR-8.
  • 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 human Ras-binding protein 66 isolated. 66kDa is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention
  • the determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0542dll was new DNA.
  • the inserted cDNA fragments contained in this clone were determined in both directions by synthesizing a series of primers.
  • the results show that the full-length cDNA contained in the 0542dll clone is 2840bp (as shown in Seq IDN0: 1), and there is a 1809bp open reading frame (0RF) from 60bp to 1868bp, which encodes a new protein (such as Seq ID NO: 2).
  • This clone pBS-0542dll was named human Ras-binding protein 66.
  • Example 2 Homologous search of cDNA clones
  • the sequence of the human Ras-binding protein 66 of the present invention and the protein sequence encoded by the same were subjected to the Blast program (Basiclocal Alignment search tool) [Altschul, SF et al. J. Mol. Biol. 1990; 215: 403-10], homology search in databases such as Genbank, Swissport.
  • the gene with the highest homology to the human Ras-binding protein 66 of the present invention is a known human Ras-binding protein SUR-8, which encodes a protein with accession number AF068920 in Genbank.
  • the protein homology results are shown in Figure 1. The two are highly homologous, with an identity of 31% and a similarity of 50%.
  • Example 3 Cloning of a gene encoding human Ras-binding protein 66 by RT-PCR
  • CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer for reverse transcription.
  • PCR amplification was performed with the following primers:
  • Primer2 5,-ATTCTGAACATGTAGTATTTATC- 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 'end reverse sequence in SEQ ID NO: 1.
  • Amplification conditions 50 mmol / L KC1, 10 mmol / L Tris-Cl, (pH 8.5), 1.5 mmol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol primers in a 50 ⁇ 1 reaction volume, 1U of Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 55 ° C 30sec; 72. C 2min.
  • ⁇ -act in 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 that of 1-2840bp shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of human Ras binding protein 66 gene expression:
  • RNA was synthesized by electrophoresis on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 mM EDTA-2.2M formaldehyde. It was then transferred to a nitrocellulose membrane.
  • the DNA probe used was the PCR amplified human Ras binding protein 66 coding region sequence (60bp to 1868bp) shown in FIG. 1.
  • a 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 (pH 7.4) -5 x SSC-5 x Denhardt's solution and 200 ⁇ g / ml salmon sperm DNA. After hybridization, place the filter at 1> SSC- Wash in 0.1% SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 5 In vitro expression, isolation and purification of recombinant human Ras-binding protein 66
  • Primer 3 5,-CATGCTAGCATGTCGCGCCTGAAGCGGATAGCG- 3, (Seq ID No: 5)
  • Primer 4 5,-CCCAAGCTTTTAAGTAGGAATTCGGTCTCTCAA- 3, (Seq ID No: 6)
  • the 5 'ends of these two primers contain Nhel and Hindlll restriction sites, respectively , followeded by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively.
  • the Nhel and Hindlll restriction sites correspond to the selectivity on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Endonuclease site.
  • PCR was performed using the pBS-0542dll plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of pBS- 0542dll plasmid, Primer-3 and Primer-4 were lOpmol and Advantage polymerase Mix (Clontech) 1 ⁇ 1, respectively. Cycle parameters: 94 ° C 20s, 60. C 30s, 68 ° C 2 min, 25 cycles. Nhel and Hindlll were used to double-digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligation product was transformed into coliform bacteria DH5oc by the calcium chloride method. After being cultured overnight in LB plates containing kanamycin (final concentration 30 g / ml), positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0542dll) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) by the calcium chloride method.
  • the host bacteria BL21 (pET-0542dll) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of lmmoi / L. Continue incubation for 5 hours. The bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. The affinity chromatography column His. Bind Quick Cartridge (product of Novagen) was used to obtain 6 histidines (6His-Tag). The purified human protein Ras-binding protein 66 was obtained.
  • the following peptides specific to human Ras-binding protein 66 were synthesized using a peptide synthesizer (product of PE): NH 2 -Met-Ser-Arg-Leu-Lys-Arg-I le-Ala-Gly-Gln-Asp-Leu- Arg-Ala-Gly-COOH (SEQ ID NO: 7).
  • the peptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is identified whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can also be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissues 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 a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern imprinting, Northern blotting, and copying methods. They all use the same basic hybridization method after fixing 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.
  • unhybridized probes are removed by a series of membrane washes.
  • This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • 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 Polysuccinic acid SEQ ID NO: 1 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 for use as hybridization probes from the polynucleotide SEQ ID NO: 1 of the present invention should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • 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 membranes nitrocellulose membranes
  • the sample membrane was placed in a plastic bag, and 3-10 mg of prehybridization solution (10xDenhardfs; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA)) was added. After the bag was sealed, it was shaken at 68 ° C for 2 hours in a water bath.
  • prehybridization solution 10xDenhardfs; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA)
  • probe 1 can be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues.
  • polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infections and immune diseases.
  • the polypeptide of the present invention can regulate the expression level of Ras protein in cells, and thus has an important role in the body, including the abnormal proliferation of tissue cells and the abnormal expression process of proteins, so that the cells can be used to control Growth, division, death, differentiation to form tissues and various life processes, and can be further used to diagnose and treat a variety of related diseases, such as: various malignant tumors and cancers, various development disorders, various immune Systemic diseases, etc.
  • the expression of this protein is related to the occurrence of various malignant tumors and cancers; therefore, the polypeptide of the present invention can be used for the diagnosis and treatment of many diseases, such as various malignancies related to it.
  • Tumors and cancers these diseases include, but are not limited to, stomach cancer, liver cancer, colorectal cancer, breast cancer, lung cancer, prostate cancer, cervical cancer, pancreatic cancer, esophageal cancer, pituitary adenoma, benign thyroid tumor, thyroid cancer, thyroid Parathyroid adenoma, parathyroid carcinoma, adrenal myeloma, pheochromocytoma, islet cell tumor, multiple endocrine gland tumor, thymus tumor, etc.
  • the human Ras-binding protein 66 of the present invention can also be used for the diagnosis and treatment of various developmental disorders related thereto, including but not limited to the following, spina bifida, craniocerebral fissure, anencephaly, brain bulge, pores Brain malformation, Down syndrome, congenital hydrocephalus, aqueduct malformation, dwarfism of cartilage hypoplasia, spinal epiphyseal dysplasia, pseudochondral dysplasia, Langer- Giedion syndrome, funnel chest, gonad hypoplasia, congenital Adrenal hyperplasia, upper urethral tract, crypt, accompanied by short stature syndromes such as Conradi and Danbolt-Closs syndrome, congenital glaucoma or cataract, congenital lens abnormality, congenital blepharoplasia, retinal dysplasia , Congenital Optic Nerve Atrophy, Congenital Sensorineural Hearing Loss, Split-hand and Split-foot Disorders, Teratology
  • the human Ras-binding protein 66 of the present invention can also be used for the diagnosis and treatment of various immune system diseases associated with its abnormal expression, including but not limited to the following, rheumatoid arthritis, chronic active hepatitis, primary Sjogren's syndrome, acute anterior uveitis, arthritis after gonococcal infection, ankylosing spondylitis, hemochromatosis, immune complex glomerulonephritis, myocarditis after gonococcal infection, systemic lupus erythematosus, rheumatoid joints Inflammation, scleroderma, polymyositis, xerostomia, Nodular polyarteritis, Wegener's granulomatosis, myasthenia gravis, Guillain-Barre syndrome, autoimmune hemolytic anemia, immune thrombocytopenic purpura, autoimmune interstitial nephritis, autoimmune gastritis, Insulin autoimmune syndrome, autoimmune thyroid disease
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human Ras-binding protein 66.
  • Agonists enhance human Ras-binding protein 66 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 human Ras-binding protein 66 can be cultured with labeled human Ras-binding protein 66 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human Ras-binding protein 66 include antibodies, compounds, receptor deletions, and analogs that have been screened. Antagonists of human Ras-binding protein 66 can bind to human Ras-binding protein 66 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions.
  • human Ras-binding protein 66 can be added to a bioanalytical assay to determine whether the compound is an antagonist by measuring the effect of the compound on the interaction between human Ras-binding protein 66 and its receptor. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to human Ras binding protein 66 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, human Ras-binding protein 66 molecules 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 against human Ras binding protein 66 epitopes. These antibodies include (but are not limited to): Doklon antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, and fragments from Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human Ras-binding protein 66 directly into immunized animals (eg, home immunity, mice, rats, etc.).
  • immunized animals eg, home immunity, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant.
  • Techniques for preparing monoclonal antibodies to human Ras-binding protein 66 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma technology, and EBV-hybridization. Tumor 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 human Ras binding protein 66.
  • Antibodies against human Ras-binding protein 66 can be used in immunohistochemical techniques to detect human Ras-binding protein 66 in biopsy specimens.
  • Monoclonal antibodies that bind to human Ras-binding protein 66 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.
  • human Ras-binding protein 66 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 the antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human Ras-binding protein 66 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human Ras binding protein 66.
  • Administration of an appropriate dose of antibody can stimulate or block the production or activity of human Ras-binding protein 66.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human Ras-binding protein 66.
  • These tests are well known in the art and include FI SH assays and radioimmunoassays.
  • the assay detected 66 human Ra s protein binding levels, it may serve to explain the importance of the human binding protein 66 R as in various diseases and for the diagnosis of human Ras binding protein 66 function disorders.
  • 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 human Ras binding protein 66 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 Ras-binding protein 66. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human Ras binding protein 66 to inhibit endogenous human Ras binding protein 66 activity.
  • a variant human Ras-binding protein 66 may be a shortened human Ras-binding protein 66 lacking a signaling domain, and although it can bind to downstream substrates, it lacks signaling activity.
  • recombinant gene therapy vectors can be used to treat diseases caused by abnormal expression or activity of human Ras-binding protein 66.
  • Virus-derived expression vectors such as retroviruses, adenoviruses, adenovirus-associated viruses, herpes simplex virus, and parvoviruses can be used to transfer polynucleotides encoding human Ras binding protein 66 into cells.
  • Methods for constructing recombinant viral vectors carrying a polynucleotide encoding human Ras-binding protein 66 can be found in existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human Ras binding protein 66 can be packaged into liposomes and transferred into cells.
  • Methods for introducing polynucleotides into tissues or cells include: Injecting polynucleotides directly into tissues in vivo Or in vitro through a vector (such as a virus, phage, or plasmid) to introduce the polynucleotide into the cell, and then transplant the cell into the body.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit human Ras binding protein 66 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 the solid-phase phosphoramidite chemical synthesis method for oligonucleotide synthesis.
  • 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 phosphorothioate or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human Ras binding protein 66 can be used for the diagnosis of diseases related to human Ras binding protein 66.
  • the polynucleotide encoding human Ras-binding protein 66 can be used to detect the expression of human Ras-binding protein 66 or the abnormal expression of human Ras-binding protein 66 in a disease state.
  • the DNA sequence encoding human Ras-binding protein 66 can be used to hybridize biopsy specimens to determine the expression of human Ras-binding protein 66.
  • 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.
  • Human Ra s-binding protein 66 specific primers can also be used to detect human Ra s-binding protein 66 transcripts by in vitro amplification of RNA-polymerase chain reaction (RT-PCR).
  • RT-PCR RNA-polymerase chain reaction
  • Detection of mutations in the human Ras binding protein 66 gene can also be used to diagnose human Ras binding protein 66-related diseases.
  • Human Ras binding protein 66 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human Ras binding protein 66 DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for labeling chromosome positions.
  • an important first step is to locate these DNA sequences on a chromosome.
  • PCR primers preferably 15-35 bp
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, 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 pre-selection of hybridization to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in a single step.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendel ian Inheritance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the difference in cDM 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 observed in any normal individual, the mutation may be the cause of the disease. Comparing diseased and diseased individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA 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 present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which reminders authorize them to be administered to humans by government agencies that manufacture, use, or sell them.
  • 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.
  • Human Ras-binding protein 66 is administered in an amount effective to treat and / or prevent a specific indication. The amount and dosage range of human Ras-binding protein 66 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.
  • Leu Lys Ser Val Pro Asp Glu lie lie Leu Leu Arg Ser Leu Glu

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Abstract

Cette invention concerne une nouvelle variante de protéine 66 humaine associée à Ras et le polynucléotide codant pour ce polypeptide ainsi que l'obtention dudit polypeptide par des méthodes recombinantes. L'invention concerne également l'utilisation de ce polypeptide dans le traitement de divers types de pathologies, dont le cancer, l'hémopathie, l'infection par le VIH, les troubles de l'immunité et les inflammations. Cette invention concerne également un antagoniste de ce polypeptide et son utilisation thérapeutique. Enfin, elle concerne le polynucléodite codant pour la protéine 66 humaine associée à Ras.
PCT/CN2000/000463 1999-11-22 2000-11-20 Nouveau polypeptide - proteine 66 humaine associee a ras, et polynucleotide codant pour ledit polypeptide WO2001038367A1 (fr)

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CN99124042A CN1296956A (zh) 1999-11-22 1999-11-22 一种新的多肽——人Ras结合蛋白66和编码这种多肽的多核苷酸
CN99124042.1 1999-11-22

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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SIEBURTH D.S., SUN Q. AND HAN M.: "Sur-8, a conserved ras-binding protein with leucine-rich repeats, positively regulates ras-mediated signaling in C. elegans", CELL, vol. 94, no. 1, July 1998 (1998-07-01), pages 119 - 130 *

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