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WO2001068875A1 - Nouveau polypeptide, proteine humaine 67 de sarcome, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, proteine humaine 67 de sarcome, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001068875A1
WO2001068875A1 PCT/CN2001/000179 CN0100179W WO0168875A1 WO 2001068875 A1 WO2001068875 A1 WO 2001068875A1 CN 0100179 W CN0100179 W CN 0100179W WO 0168875 A1 WO0168875 A1 WO 0168875A1
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Prior art keywords
polypeptide
polynucleotide
protein
human sarcoma
sarcoma protein
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PCT/CN2001/000179
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc.
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Application filed by Shanghai Biowindow Gene Development Inc. filed Critical Shanghai Biowindow Gene Development Inc.
Priority to AU39117/01A priority Critical patent/AU3911701A/en
Publication of WO2001068875A1 publication Critical patent/WO2001068875A1/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
    • C07K14/82—Translation products from oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, human sarcoma protein 67, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide.
  • the growth, differentiation, and proliferation of cells constitute an important part of the cell's life history. Problems in any of these sections will cause changes in the cell's survival status, which will affect the health of the entire body. Therefore, genes that play a regulatory role in any of these links are particularly important.
  • the myb gene plays a regulatory role in cell growth, differentiation, and death, and is a very important gene.
  • the myb gene was the earliest oncogene V- myb found in chickens, and its corresponding gene in normal cells was c-myb. Later, several new myb genes were discovered, A-myb and B-myb. They all contain three similar structures: an N-terminal D-binding region, a centrally-active transcriptional structure, and a C-terminal para-regulatory region.
  • myb gene Structures similar to the myb gene are found in many organisms, such as plants, yeast, and mold.
  • the myb gene is generally expressed higher in proliferating cells. At the end of differentiation and in cells that exit the cell cycle, the rayb gene is turned off.
  • B-myb is expressed in the whole process from implantation to maturation of fertilized eggs, while C-myb is highly expressed in hematopoietic organs, smooth muscles and rectal mucosa.
  • A-myb is widely expressed in the actively dividing tissues during embryonic development But after birth, it is expressed only in lymph and reproductive tissues.
  • the myb gene also plays an important role in the regulation of cell differentiation.
  • C-Myb is negatively regulated during hematopoietic cell differentiation and maturation. If c-Myb is overexpressed, the differentiation of immature bone marrow and red blood cell lines is inhibited.
  • a possible mechanism for c-Myb to regulate cell differentiation is: c-Myb regulates a complex gene group, which then plays an important regulatory role in the cell differentiation process.
  • Myb protein is also a regulator of survival and death of bone marrow cells and T cells during development. Studies have found that turning off the Myb-E t s fusion protein in chicken myeloblasts results in cell death. Overexpression of c-Myb protein can protect CTLL-2 T cells from apoptosis due to interleukins, suggesting that at least T cells, c-Myb has an important anti-apoptotic effect.
  • Over-expression of Myb protein will cause cells to proliferate, inhibit cell differentiation and death, and produce tumors. For example, lymphoma, leukemia, etc. Overexpression of c-Myb in hematopoietic cells of the liver can lead to liver cancer. In addition, the absence of Myb or a low expression level will lead to a reduction in the rate of cell proliferation and development of various developmental diseases, as well as a decrease in the buffer capacity of cells to resist death from external influences.
  • the expression profile of the polypeptide of the present invention is very similar to the expression profile of human sarcoma protein 20, so their functions may also be similar.
  • the invention is named human sarcoma protein 67.
  • human sarcoma protein 67 protein plays an important role in regulating important functions of the body, such as cell growth and differentiation, and it is believed that a large number of proteins are involved in these regulatory processes. Therefore, there has been a need in the art to identify more human sarcomas involved in these processes. Protein 67 protein, particularly the amino acid sequence of this protein. Isolation of the new human sarcoma protein 67 protein encoding gene 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 developing diagnostic and / or therapeutic drugs, so isolating its coding DNA is important. Object of the invention
  • Another object of the present 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 human sarcoma protein 67.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding human sarcoma protein 67.
  • Another object of the present invention is to provide a method for producing human sarcoma protein 67.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to the abnormality of human sarcoma protein 67. Summary of invention
  • the invention relates to an isolated polypeptide, which is of human origin, and which comprises: SEQ ID No. 2 Amino acid sequence of a polypeptide, 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 431-2263 in SEQ ID NO: 1; and (b) a sequence having 1-2531 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 the activity of human sarcoma protein 67 protein, which comprises utilizing the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the present invention also relates to a method for detecting a disease or disease susceptibility associated with abnormal expression of human sarcoma protein 67 protein in vitro, which comprises detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, or detecting a mutation in a biological sample.
  • the amount or biological activity of a polypeptide of the invention comprises detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, or detecting a mutation 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 the use of the polypeptides and / or polynucleotides of the present invention in the preparation of a medicament for the treatment of malignant tumors, developmental disorders and immune diseases or other diseases caused by abnormal expression of human sarcoma protein 67.
  • FIG. 1 is a comparison diagram of gene chip expression profiles of human sarcoma protein 67 and human sarcoma protein 20 of the present invention.
  • the upper graph is a graph of the expression profile of human sarcoma protein 67
  • the lower graph is the graph of the expression profile of human sarcoma protein 20.
  • Figure 2 is a polyacrylamide gel electrophoresis image (SDS-PAGE) of isolated human sarcoma protein 67.
  • 67kDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band. Summary of the invention
  • 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 DNA or RNA, 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, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to 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 a change 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 sarcoma protein 67, causes a change in the protein to regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that binds human sarcoma protein 67.
  • Antagonist refers to a molecule that, when combined with human sarcoma protein 67, can block or regulate the biological or immunological activity of human sarcoma protein 67.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind human sarcoma protein 67.
  • Regular refers to a change in the function of human sarcoma protein 67, including an increase or decrease in protein activity, a change in binding properties, and any other biological, functional, or immune properties of human sarcoma protein 67 Change.
  • 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 sarcoma protein 67 using standard protein purification techniques.
  • Substantially pure Human sarcoma protein 67 can generate a single main band on a non-reducing polyacrylamide gel.
  • the purity of human sarcoma protein 67 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. This inhibition of hybridization can be detected by performing hybridization (Southern imprinting or Nor thern 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 the same or similar in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, such as through the MEGALIGN program
  • the MEGALIGN program can compare two or more sequences (H i gg i ns, D. G. and
  • the percent identity between nucleic acid sequences can also be determined by the Cluster method or by methods known in the art such as Jotun He in (He in L, (1990) Methods in enzymo l ogy 183: 625-645). "Sexuality” refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • negatively charged amino acids may include aspartic acid and glutamic acid; Positively charged amino acids can include lysine and arginine; amino acids with similarly charged head groups that have similar hydrophilicity can include leucine, isoleucine, and valine; glycine and alanine Acids; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular D or RM sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to the "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? It can specifically bind to the epitope of human sarcoma protein 67.
  • 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 matter 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 vector, or such a polynucleotide or polypeptide may be part of a 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 in the natural state .
  • isolated human sarcoma protein 67 means that human sarcoma protein 67 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 sarcoma protein 67 using standard protein purification techniques. Substantially pure polypeptides produce a single main band on non-reducing polyacrylamide gels. The purity of the human sarcoma protein 67 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human sarcoma protein 67, which basically consists of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptide of the present invention may be a naturally purified product, or a chemically synthesized product, or may be produced from a prokaryotic or eukaryotic host (for example, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant technology. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of human sarcoma protein 67.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human sarcoma protein 67 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may So: (I) a type in which one or more amino acid residues are replaced by conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substituted amino acid may or may not be a genetic codon Encoded; or ( ⁇ ) such a type in which a group on one or more amino acid residues is substituted with another group to include a substituent; or (in) such a type in which the mature polypeptide and another compound (Such as a compound that extends the half-life of a polypeptide, such as polyethylene glycol); or (IV) a polypeptide sequence in which an additional amino acid sequence is fused into a mature polypeptide (such as a leader sequence or a secreted sequence or used to purify this) The sequence of the polypeptide or protease). As set forth herein, such fragments, and their 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 25 31 bases, and its open reading frame 4 31-226 3 encodes 610 amino acids. According to the comparison of gene chip expression profiles, it was found that this peptide has a similar expression profile with human sarcoma protein 20, and it can be concluded that human sarcoma protein 67 has a similar function to human sarcoma protein 20.
  • 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 the 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 D NO: 2 but different from the coding region sequence shown in SEQ ID D 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 present invention also relates to a polynucleotide that hybridizes to a sequence described above 50% less, preferably 70% identity).
  • the invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the 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, 6 (TC; or (2) added during hybridization) Use a denaturant, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42'C, etc .; or (3) the identity between the two sequences is at least 95% Above, it is more preferable that the hybridization occurs at 97% or more. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • 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 sarcoma protein 67.
  • 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 sarcoma protein 67 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) separating the double-stranded DNA sequence from the DM of the genome; 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 separation of cDM sequences.
  • the standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature techniques for mRNA extraction, and kits are also commercially available (Qiagene).
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When 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-RM hybridization; (2) the appearance or loss of marker gene function; (3) measuring the level of human sarcoma protein 67 transcripts; (4) passing Immunological techniques or assays for biological activity to detect gene-expressed protein products. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 Nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is usually a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DM 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 of human sarcoma protein 67 gene expression.
  • ELISA enzyme-linked immunosorbent assay
  • 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-cDM terminal rapid amplification method
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be measured by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDM sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising a polynucleotide of the present invention, and a host cell genetically engineered using the vector of the present invention or directly using a human sarcoma protein 67 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology.
  • a polynucleotide sequence encoding human sarcoma protein 67 may be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors expressed in bacteria (Rosenberg, et al.
  • pMSXND expression vectors expressed in mammalian cells Lee and Nathans, J Bio Chem. 263: 3521, 1988
  • baculovirus-derived vectors expressed in insect cells in short, as long as it can be replicated and stabilized in the host, any plasmid and vector can be used to construct recombinant expression vectors.
  • 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 DM sequences encoding human sarcoma protein 67 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 guide mRNA synthesis. Representative examples of these promoters are: l ac 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 and adenovirus enhancers on the late side of the origin of replication.
  • 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 human sarcoma protein 67 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.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells insect cells
  • fly S 2 or Sf 9 animal cells
  • animal cells such as CH0, COS or Bowes s melanoma cells Wait.
  • Transformation of a host cell with a DM sequence according to the present invention or a recombinant vector containing the DNA sequence can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DM may be harvested after exponential growth phase, treated with CaC l 2 method used in steps well known in the art. The alternative is to use MgC l 2 .
  • 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 liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human sarcoma protein 67 (Scence, 1 984; 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
  • polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, such as malignant tumors, developmental disorders and immune diseases.
  • the myb gene plays a regulatory role in cell growth, differentiation, and death, and can be used to treat and prevent various developmental disorders and cancers.
  • the polypeptide of the present invention or a fragment thereof can be used to treat or prevent diseases caused by developmental disorders, including but not limited to: spina bifida, craniocerebral fissure, anencephaly, cerebral bulge, foramen forebral malformation, Down syndrome, congenital Hydrocephalus, aqueduct malformation, cartilage hypoplasia, dwarfism, spinal epiphyseal dysplasia, pseudochondral dysplasia, Langer-G i ed i on syndrome, funnel chest, gonad hypoplasia, congenital adrenal hyperplasia, Upper urethral fissure, cryptorchidism, short stature syndrome such as Conrad i syndrome and Danbo l tC los s syndrome, congenital glaucoma or cataract, congenital lens position abnormality, congenital blepharoplasia, retinal dysplasia , Congenital Optic Nerve Atrophy, Congenital Sensorineural Hearing Loss, Split
  • polypeptides or fragments thereof of the present invention can also be used to treat or prevent cancer. Including but not limited to: Common tumors of each system are as follows:
  • Digestive system tumors salivary gland tumors, esophageal cancer, esophageal leiomyosarcoma, primary esophageal small cell carcinoma, gastric cancer, gastric malignant lymphoma, gastric carcinoid, colorectal cancer, colon cancer, intestinal malignant lymphoma, primary liver cancer, Hepatoblastoma, primary gallbladder cancer, pancreatic cancer
  • Hematological and Lymphatic Tumors Acute Leukemia, Chronic Myeloid Leukemia, Chronic Lymphocytic Leukemia Hematopathy, malignant lymphoma (such as lymphatic reticulum, malignant lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, etc.), malignant histiocytosis
  • Nervous system tumors astrocytoma, ependymal tumor, medulloblastoma, meningiomas, glioblastoma, acoustic neuroma, angiogenic tumor, pituitary adenoma, craniopharyngioma
  • osteoid osteoma osteochondroma, chondroma, osteoblastoma, chondroblastoma, etc.
  • malignant bone tumors such as giant cell tumor of bone, osteosarcoma, chondrosarcoma, Ewing's sarcoma, myeloma urinary Reproductive system tumors: Benign tumors such as renal cortical tubular adenoma, eosinophil adenoma, juxtaglomerular cell tumor, polycystic kidney tumor, seminoma, teratoma, testicular stromal tumor, endometrium Interstitial tumor, hydatidiform mole, ovarian tumor, breast fibroma, malignant tumors such as renal cell carcinoma, renal sarcomatoid carcinoma, papillary renal cell carcinoma, nephroblastoma, prostate cancer, testicular tumor chorionic carcinoma, epididymal cancer, child Cervical cancer, endometrial cancer,
  • Soft tissue tumors fibroma, fibrosarcoma, fibromatosis, lipoma, liposarcoma, leiomyoma, leiomyosarcoma, rhabdomyosarcoma, rhabdomyosarcoma, synovial tissue tumor, hemangioma, intramuscular hemangioma, blood vessels Globuloma, hemangioendothelial sarcoma, lymphangioma, lymphangiomyoma, lymphatic endothelial sarcoma, histiocytoma, malignant fibrous histiocytoma, soft tissue acinar sarcoma, clear cell sarcoma, myxoma, extraosseous Ewing's sarcoma, Soft tissue osteosarcoma, soft tissue chondrosarcoma, mesothelioma, epithelioid sarcoma, schwannomas, neurofibromas, malignant
  • Skin malignancies dermal Mike cell tumor, Kaposi sarcoma, melanoma
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human sarcoma protein 67.
  • Agonists enhance human sarcoma protein 67 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 sarcoma protein 67 can be cultured with labeled human sarcoma protein 67 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human sarcoma protein 67 include antibodies, compounds, receptor deletions and analogs. Antagonists of human sarcoma protein 67 can bind to human sarcoma protein 67 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 sarcoma protein 67 When screening compounds as antagonists, human sarcoma protein 67 can be added to bioanalytical assays to determine the effect of the compound on the interaction between human sarcoma protein 67 and its receptor 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. Polypeptide molecules capable of binding to human sarcoma protein 67 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. In screening, the human sarcoma protein 67 molecule should generally be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against human sarcoma protein 67 epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments generated from Fab expression libraries.
  • polyclonal antibodies can be obtained by direct injection of human sarcoma protein 67 into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant.
  • Techniques for preparing monoclonal antibodies to human sarcoma protein 67 include, but are not limited to, hybridoma technology (Kohler and Milstei n. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma technology , EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions and non-human-derived variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851).
  • the existing technology for producing single chain antibodies U.S. Pat No. 4946778, can also be used to produce single chain antibodies against human sarcoma protein 67.
  • Anti-human sarcoma protein 67 antibodies can be used in immunohistochemical techniques to detect human sarcoma protein 67 in biopsy specimens.
  • Monoclonal antibodies that bind to human sarcoma protein 67 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 sarcoma protein 67 high affinity monoclonal antibodies can covalently bind to bacterial or phytotoxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human sarcoma 67 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human sarcoma protein 67.
  • Administration of an appropriate dose of antibody can stimulate or block the production or activity of human sarcoma protein 67.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human sarcoma protein 67 levels. These tests are well known in the art and include FI SH assays and radioimmunoassays.
  • the level of human sarcoma protein 67 detected in the test can be used to explain the importance of human sarcoma protein 67 in various diseases and to diagnose diseases in which human sarcoma protein 67 plays a role.
  • the polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzyme, and one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • the polynucleotide encoding human sarcoma protein 67 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 sarcoma protein 67.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human sarcoma protein 67 to inhibit endogenous human sarcoma protein 67 activity.
  • a mutated human sarcoma protein 67 may be a shortened human sarcoma protein 67 lacking a signaling domain. 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 sarcoma protein 67.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus and the like can be used to transfer a polynucleotide encoding human sarcoma protein 67 into cells.
  • Methods for constructing recombinant viral vectors carrying a polynucleotide encoding human sarcoma protein 67 can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human sarcoma protein 67 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 sarcoma protein 67 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 is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RM, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technology, such as solid-phase phosphate amide chemical synthesis to synthesize oligonucleotides.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA.
  • This DNA sequence has been integrated downstream of the RNA polymerase promoter of the vector.
  • it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the phosphorothioate or peptide bond instead of the phosphodiester bond is used for the ribonucleoside linkage.
  • the polynucleotide encoding human sarcoma protein 67 can be used for the diagnosis of diseases related to human sarcoma protein 67.
  • the polynucleotide encoding human sarcoma protein 67 can be used to detect the expression of human sarcoma protein 67 or the abnormal expression of human sarcoma protein 67 in a disease state.
  • the DNA sequence encoding human sarcoma protein 67 can be used to hybridize biopsy specimens to determine the expression of human sarcoma protein 67.
  • Hybridization techniques include Sout hern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • Part or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray (Microray) or a DNA chip (also known as a "gene chip") for analyzing differential expression analysis of genes and genes in tissues. diagnosis.
  • a microarray Microray
  • a DNA chip also known as a "gene chip”
  • RT-PCR RNA-polymerase chain reaction
  • amplification of the primers can also be used to detect human sarcoma protein 67 transcripts.
  • Human sarcoma protein 67 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human sarcoma protein 67 DNA sequence. Mutations can be detected using existing techniques such as Southern imprinting, DNA sequence analysis, PCR, and in situ hybridization. In addition, mutations may affect protein expression. Therefore, Northern 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 located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing 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 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 one step.
  • FISH Fluorescent in situ hybridization
  • the differences in cDNA or genomic sequences between the affected and unaffected individuals need to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing diseased and unaffected individuals usually involves first looking for structural changes in the chromosome, such as defects visible at the chromosomal level or detectable by cDNA sequence-based PCR Missing or transposing. 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 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.
  • Human sarcoma protein 67 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human sarcoma protein 67 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. 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 Isolation Kit (product of Qiegene). 2ug poly (A) mRNA forms CDM by reverse transcription.
  • the Smart cDNA cloning kit purchased from Clontech was used to insert the cDNA fragments into the multicloning site of pBSK (+) vector (Clontech) to transform DH5 oc.
  • the bacteria formed a CDM library.
  • the sequences at the 5 'and 3' ends of all clones were determined using Dye termina te cyc le react ion sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer).
  • the determined cDNA sequence was compared with the existing public DM sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0404A12 was new DNA. Insert a cDNA fragment into the clone by synthesizing a series of primers Segments are measured in both directions.
  • CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer for reverse transcription reaction. After purification using Qiagene's kit, the following primers were used for PCR amplification:
  • Primerl 5'- GAAAGAGGCTAAATTTTTCTTGAC -3, (SEQ ID NO: 3)
  • Primer2 5'- CCACAGCACCTTCCATTTTATTGT -3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification reaction conditions reaction volume containing 50 ⁇ 1 of 50mmol / L KC1, 10 bandit ol / L Tris-HCl, pH8.5 , 1.5mmol / L MgCl 2, 200 ⁇ ⁇ 1 / ⁇ dNTP, lOpmol primer, 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.
  • P-actin was set as a positive control and template blank was set as a negative control.
  • the amplified product was purified using a QIAGEN kit and ligated to a pCR vector using a TA cloning kit (Invitrogen). DM sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as l-2531bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human sarcoma protein 67 gene expression
  • This method involves acid guanidinium thiocyanate phenol-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ), Mix and centrifuge. 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 (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 ( pH7.4)-5 x SSC-5 ⁇ Denhardt's solution and 20 ( ⁇ g / ml salmon sperm DNA. After hybridization, the filter was placed in 1 ⁇ SSC- 0.1% SDS in Wash at 55 ° C for 30min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 4 In vitro expression, isolation and purification of recombinant human sarcoma protein 67
  • Primer3 5'-CCCCATATGATGGACATCAAAGGCCAGTTCTGG-3 '(Seq ID No: 5)
  • Priraer4 5'-CCCGAATTCTCAGTTTGTGTAAACCTGAGTTCC-3' (Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and EcoRI restriction sites, respectively.
  • the coding sequences of the 5 'and 3' ends of the gene of interest are followed, respectively.
  • the Ndel and EcoRI restriction sites correspond to the selectivity within the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Digestion site.
  • PCR reaction was performed using the PBS-0404A12 plasmid containing the full-length target gene as a template.
  • PCR reaction conditions were: 1 in a total volume of 50 ⁇ plasmid pBS- 0404A12 containing 10pg, primer Primer- 3 and Pr imer- 4 are lOpmol, Advantage polymerase Mix (Clontech Products) 1 ⁇ 1.
  • Cycle parameters 94. C 20s, 60. C 30s, 68. C 2 min, a total of 25 cycles.
  • Ndel 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 E.
  • 6His-Tag 6 histidines
  • 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 determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in 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, Nor thern blotting, and copying methods. They all use the same steps to fix the polynucleotide sample to be tested on the filter and then hybridize.
  • 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) 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
  • 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 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 mutation sequence (41Nt) of the gene fragment or its complementary fragment of SEQ ID NO: 1:
  • 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 32 P-Probe (the second peak is free ⁇ - 32 P-dATP) is prepared.
  • the sample membrane was placed in a plastic bag, and 3 to 10 mg of prehybridization solution (10xDenhardfs; 6xSSC, 0.1 mg / ml CT DM (calf thymus DNA)) was added. After sealing the mouth of the bag, shake at 68 ° C for 2 hours.
  • prehybridization solution 10xDenhardfs; 6xSSC, 0.1 mg / ml CT DM (calf thymus DNA)
  • Gene chip or gene microarray is a new technology currently being developed by many national laboratories and large pharmaceutical companies.
  • 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 a target DM for gene chip technology for high-throughput research of new gene functions; searching for and screening new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • the specific method steps have been reported in the literature. For example, see the literature DeRi si, JL, Lyer, V. & Brown, P. 0. (1997) Sc ience 278, 680-686. And the literature Hel le, RA, Schema, M., Cha i, A., Sha lom, D., (1997) PNAS 94: 2150-2155.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as the target DM, including the polynucleotide of the present invention. They were amplified by PCR respectively. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and spotted on a glass medium using a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between the points is 280 ⁇ . 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:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and the mRNA was purified using Oligotex raRNA Mid i Ki t (purchased from Qi aGen).
  • 1 J the fluorescent reagent Cy3dUTP (5-Amino-propargyl-2 '-deoxyur idine 5--tr iphate coupled to Cy3 fluorescent dye, purchased from Amersham Phamacia Biotech) labeled niRNA of human mixed tissue, using a fluorescent reagent Cy5dUTP (5- Amino-propargy 2'-deoxyuridine 5--triphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech The company) labeled the mRNA of specific tissues (or stimulated cell lines) of the body and prepared probes after purification.
  • Cy3dUTP 5-Amino-propargyl-2 '-deoxyur idine 5--tr iphate coupled to Cy3 fluorescent dye, purchased
  • the probes from the two types of tissues and the chips were hybridized in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, washed with a washing solution (1 x SSC, 0.2 SDS) at room temperature and scanned with ScanArray 3000
  • the instrument purchased from General Scanning Company, USA was used for scanning.
  • the scanned image was analyzed and processed with Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, Arsenic stimulated the L02 cell line and prostate tissue for 1 hour. Based on these 13 Cy3 / Cy5 ratios, draw a bar graph ( Figure 1). It can be seen from the figure that the expression profiles of human sarcoma protein 67 and human sarcoma protein 20 according to the present invention are very similar.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine humaine 67 de sarcome, et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, des troubles du développement et des maladies immunitaires. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la protéine humaine 67 de sarcome.
PCT/CN2001/000179 2000-03-15 2001-02-26 Nouveau polypeptide, proteine humaine 67 de sarcome, et polynucleotide codant pour ce polypeptide WO2001068875A1 (fr)

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CN 00114920 CN1313334A (zh) 2000-03-15 2000-03-15 一种新的多肽——人肉瘤蛋白67和编码这种多肽的多核苷酸

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5411860A (en) * 1992-04-07 1995-05-02 The Johns Hopkins University Amplification of human MDM2 gene in human tumors
US5861240A (en) * 1996-02-28 1999-01-19 The Regents Of The University Of California Isolated human herpesvirus type 8 sequences and uses thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5411860A (en) * 1992-04-07 1995-05-02 The Johns Hopkins University Amplification of human MDM2 gene in human tumors
US5861240A (en) * 1996-02-28 1999-01-19 The Regents Of The University Of California Isolated human herpesvirus type 8 sequences and uses thereof

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