WO2001032698A1 - Nouveau polypeptide, proteine humaine 16 associee aux maladies auto-immunes, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, proteine humaine 16 associee aux maladies auto-immunes, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001032698A1
WO2001032698A1 PCT/CN2000/000405 CN0000405W WO0132698A1 WO 2001032698 A1 WO2001032698 A1 WO 2001032698A1 CN 0000405 W CN0000405 W CN 0000405W WO 0132698 A1 WO0132698 A1 WO 0132698A1
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Prior art keywords
polypeptide
polynucleotide
autoimmune disease
human autoimmune
related protein
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PCT/CN2000/000405
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Bio Door Gene Technology Ltd.
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Priority to AU12660/01A priority Critical patent/AU1266001A/en
Publication of WO2001032698A1 publication Critical patent/WO2001032698A1/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
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide—human autoimmune disease-related protein 16 and a polynucleotide sequence encoding the polypeptide. The invention also relates to methods and applications for preparing such polynucleotides and polypeptides.
  • snRNP is required for the splicing and splicing of mRNA precursors.
  • SnRNP is called a nuclear small molecule ribonucleic acid protein, which is formed by the combination of snRNA (a class of small molecular weight RNA present in eukaryotic cells) and protein. of.
  • s nRNA contains a large amount of uridine, it is also called U-snRNA.
  • SnRNP exists in almost all spinal impeller cells, and six U-snRNPs such as Ul, U2, U3, U4, U5, and U6 have been found.
  • the splicing of the raRNA precursor s is more complicated and must be completed by a spliceo composed of multiple U-snRNPs. In this process, different kinds of snRNPs play different roles.
  • the snRNP mentioned in the present invention is U2-snRNP, and its main role in the splicing process is to recognize and bind the branch points of the mRNA precursor.
  • the U2- snRNP cofactor is a heterodimer: the large subunit has a molecular weight of 65kD and the small subunit has a molecular weight of 35kD.
  • the small subunit U2AF35 has degenerate arginine, serine-rich regions, and a carboxy-terminal RNA recognition mot if f region.
  • U2AF35 plays a role not only in constitutive splicing, but also in enhancer-dependent splicing. It acts as a bridge between the large subunit U2AF65 and the enhancer complex so that U2AF65 binds to adjacent introns.
  • polypeptide of the present invention was presumed to be identified as a human autoimmune disease-related protein 16.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a human autoimmune disease-related protein 16.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human autoimmune disease related protein 16.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the polypeptide of the present invention, human autoimmune disease-related protein 16.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities in human autoimmune disease-related protein 16.
  • a novel isolated human autoimmune disease-related protein 16 is provided.
  • the polypeptide is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID NO: 2, or a conservative variant polypeptide thereof, Or its active fragment, or its active derivative, analog.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • a polynucleotide encoding these isolated polypeptides, the polynucleotide comprising a nucleotide sequence having at least 66 nucleotides with a nucleotide sequence selected from the group consisting of % Identity: (a) a polynucleotide encoding the aforementioned human autoimmune disease-related protein 16; (b) a polynucleotide complementary to the polynucleotide (a).
  • the polynucleotide encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 2.
  • sequence of the polynucleotide is one selected from the group consisting of: (a) having SEQ ID NO: 1 A sequence of positions 3 to 682-111; and (b) a sequence of positions 1-1174 of SEQ ID NO: 1.
  • Fig. 1 is a comparison diagram of amino acid sequence homology of the inventor's autoimmune disease-related protein 16 and human U2-snRNP helper subunit.
  • the upper sequence is human autoimmune disease-related protein 16, and the lower sequence is human U2-snRNP helper subunit.
  • 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 isolated human autoimmune disease-related protein 16. 16kDa is the molecular weight of the protein. The arrow indicates the isolated protein band.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state. .
  • isolated human autoimmune disease-related protein 16 refers to human autoimmune disease-related protein 16 that is substantially free of other proteins, lipids, sugars, or other substances that are naturally associated with it. Those skilled in the art can purify human autoimmune disease-related proteins 16 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human autoimmune disease-related protein 16 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human autoimmune disease-related protein 16, 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 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.
  • polypeptides of the invention may be glycosylated, or they may be non-glycosylated.
  • the polypeptides of the invention may also include or exclude the initial methionine residue.
  • the present invention also includes fragments, derivatives, and analogs of human autoimmune disease-related protein 16.
  • fragments As used in the present invention, the terms “fragments”, “derivatives”, and “analogs” refer to humans who substantially retain the present invention Immune disease-related protein 16 A polypeptide with the same biological function or activity.
  • Polypeptides of the invention may be: (I) a type in which one or more amino acid residues are replaced with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substituted amino acid may be It may or may not be encoded by a genetic code; or ( ⁇ ) such a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or ( ⁇ ⁇ ) such a type Wherein the mature polypeptide is fused to another compound (such as a compound that prolongs the half-life of the 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 Sequences or secreted sequences or sequences used to purify this polypeptide or protease sequences) As set forth herein, such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 1174 bases in length and its open reading frame (682-1113) encodes 143 amino acids.
  • the polypeptide has 66% homology with the human U2- S n RNP helper subunit, and it can be inferred that the human autoimmune disease-related protein 16 has the human U2-S HRNP helper subunit. Similar structure and function.
  • 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 NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be days Naturally occurring allelic 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 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) L ° / ⁇ Denaturing agents are added during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1 ° /.
  • Hybridization occurs only when the identity between the two sequences is at least 95% or more, and more preferably 97% or more.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nuclei. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding human autoimmune disease-associated protein 16.
  • 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 autoimmune disease-related protein 16 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 DM sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice.
  • the more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • the construction of cDNA library is also a common method (Sambrook, eta l., Molecular Cloning, A Labora tory Manua l, Cold Spr ing Harbor Labora tory. New York, 1989) 0
  • 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): (1) DM-DNA or DNA-RNA hybridization; (2) the presence or loss of marker gene function; (3) determination of the level of transcripts of human autoimmune disease-related protein 16; ( 4) Detecting gene-expressed protein products by immunological techniques or by measuring biological activity. 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 DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of human autoimmune disease-related protein 16 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • a method using DNA technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-cD terminal rapid amplification method
  • the primers used for PCR may be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be measured by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising a polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a human autoimmune disease-associated protein 16 coding sequence, and a recombinant technology to produce a polypeptide of the present invention. method.
  • a polynucleotide sequence encoding a human autoimmune disease-related protein 16 may be inserted into a carrier To construct 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.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human autoimmune disease-related protein 16 and appropriate transcription / translation regulatory elements. These methods include in vitro recombination 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 the expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. 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. Examples include 100 to 270 base pair SV40 enhancers at the late side of the origin of replication, polyoma enhancers and adenovirus enhancers at 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 a human autoimmune disease-related protein 16 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a gene containing the polynucleotide or the recombinant vector.
  • Genetically engineered host cells refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote, such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Alternatively, M g Cl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human autoimmune disease-related protein 16 (Sc ience, 1984; 224: 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid
  • the polypeptides of the present invention can be directly used in the treatment of diseases.
  • the invention also provides screening compounds to identify those who increase (agonist) or suppress (antagonist) the human immune system.
  • Method for Plague Disease-Related Protein 16 Agonists enhance human autoimmune disease-related protein 16 to stimulate cell proliferation and other biological functions, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • mammalian cells or expression can be expressed in the presence of drugs
  • Membrane preparations of human autoimmune disease-related protein 16 are cultured with labeled human autoimmune disease-related protein 16. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human autoimmune disease-related protein 16 include selected antibodies, compounds, receptor deletions, and the like. Antagonists of human autoimmune disease-related protein 16 can bind to human autoimmune disease-related protein 16 and eliminate it. The function either inhibits the production of the polypeptide or binds to the active site of the polypeptide so that the polypeptide cannot perform a biological function.
  • human autoimmune disease-related protein 16 can be added to bioanalytical assays to determine whether the compound is a compound by measuring the effect of the compound on the interaction between human autoimmune disease-related protein 16 and its receptor. Antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to human autoimmune disease related protein 16 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 16 molecules of human autoimmune disease-related proteins should generally be labeled.
  • the present invention provides methods for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies may be polyclonal antibodies or monoclonal antibodies.
  • the present invention also provides antibodies against human 16 autoantibodies related protein 16 epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human autoimmune disease-related protein 16 directly 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 Agent.
  • Techniques for preparing monoclonal antibodies to human autoimmune disease-related protein 16 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridization Tumor technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions to non-human variable regions can be produced using existing techniques (Morr et al, PNAS, 1985, 81: 6851).
  • Monoclonal antibodies that bind to human autoimmune disease-related protein 16 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 a toxin that targets a particular part of the body.
  • human autoimmune disease-related proteins 16 High-affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human autoimmune disease-related protein 16 positive cell.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human autoimmune disease-related protein 16.
  • Administration of appropriate doses of antibodies can stimulate or block the production or activity of human autoimmune disease-related protein 16.
  • the invention also relates to diagnostic test methods for quantitative and localized detection of human autoimmune disease-related protein 16 levels. These tests are well known in the art and include FISH assays and radioimmunoassays. The level of human autoimmune disease-related protein 16 detected in the test can be used to explain the importance of human autoimmune disease-related protein 16 in various diseases and to diagnose diseases in which human autoimmune disease-related protein 16 plays a role.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • Polynucleotides encoding human autoimmune disease-related protein 16 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 autoimmune disease-related protein 16.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant human autoimmune disease-related protein 16 to inhibit endogenous human autoimmune disease-related protein 16 activity.
  • a mutated human autoimmune disease-related protein 16 may be a shortened human autoimmune disease-related protein 16 that lacks a signaling domain. Although it can bind to downstream substrates, it lacks signaling activity.
  • the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of human autoimmune disease-related protein 16.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used for A polynucleotide encoding human autoimmune disease-related protein 16 is transferred into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a human autoimmune disease-related protein 16 can be found in the existing literature (Sambrook, eta l.).
  • a recombinant polynucleotide encoding human autoimmune disease-related protein 16 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 etc.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DM
  • ribozymes that inhibit human autoimmune disease-related protein 16 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, DM, and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as solid-phase phosphoramidite chemical synthesis to synthesize oligonucleotides.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA.
  • This DNA sequence has been integrated downstream of the RM polymerase promoter of the vector.
  • it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human autoimmune disease-related protein 16 can be used for diagnosis of diseases related to human autoimmune disease-related protein 16.
  • the polynucleotide encoding human autoimmune disease-related protein 16 can be used to detect the expression of human autoimmune disease-related protein 16 or the abnormal expression of human autoimmune disease-related protein 16 in a disease state.
  • the D sequence encoding human autoimmune disease-related protein 16 can be used to hybridize biopsy specimens to determine the expression of human autoimmune disease-related protein 16.
  • Hybridization techniques include Sou thern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are all mature and open technologies, and related kits are commercially available.
  • a part or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray (Microray) or a DNA chip (also known as a "gene chip"), and used to analyze differential expression analysis and gene diagnosis of genes in tissues.
  • a microarray Microray
  • a DNA chip also known as a "gene chip”
  • Human autoimmune disease-related protein 16 specific primers for RNA-polymerase chain reaction (RT-PCR) amplification in vitro can also detect human autoimmune disease-related protein 16 transcription products.
  • Detection of human autoimmune disease-related protein 16 gene mutations can also be used to diagnose human autoimmune disease-related protein 16-related diseases.
  • Mutated forms of human autoimmune disease-associated protein 16 include Often the wild-type human autoimmune disease-related protein 16D sequence is compared to point mutations, translocations, deletions, recombination, and any other abnormalities. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect the expression of proteins. Therefore, Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, the specific loci of each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) can be used to mark chromosome locations. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these D sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells that contain the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention by a similar method, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones to metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckus ck, Mende l ian Inher i tance in Man (available online with Johns Hopk ins University Welch Med i cal Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the differences in cDNA or genomic sequences between the affected and unaffected individuals need to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, as seen at the chromosomal level or detectable by cDNA sequence-based PCR Deletion or translocation. 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 suitable pharmaceutical carriers.
  • suitable pharmaceutical carriers can be water, glucose, ethanol, salts, buffers, glycerol and A combination thereof.
  • the composition comprises a safe and effective amount of a polypeptide or an antagonist and a carrier and an excipient 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 polypeptide of the present 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 autoimmune disease-related protein 16 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human autoimmune disease-related protein 16 administered to a patient will depend on many factors, such as administration Method, the health condition of the person to be treated, and the judgment of the diagnosis doctor.
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) raRNA was isolated from total RNA using Quik mRNA Isolat ion Kit (product of Qiegene). 2ug poly (A) mRNA was reverse transcribed to form CDM.
  • a Smart cDM cloning kit purchased from Clontech was used to insert the cDNA fragment into the multicloning site of the pBSK (+) vector (Clontech) to transform DH5 ct to form a cDNA library.
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 Automatic sequencer Perkin-Elmer
  • the determined cDNA sequences were compared with the existing public D sequence database (Genebank), and the cD sequence of one of the clones 0480G04 was found For new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • the sequence of the human autoimmune disease-related protein 16 and the encoded protein sequence of the present invention were analyzed using the Blas t program (Basic loca l al ignment search tool) [Al schul, SF et al. J. Mol. Biol. 1990; 215: 403-10], homology search in databases such as Genbank, Swiss sport, etc.
  • the gene with the highest homology to the human autoimmune disease-related protein 16 of the present invention is a known human U2 snRNP helper Subunit, the accession number of the encoded protein in Genbank is M96982.
  • the protein homology results are shown in Figure 1. The two are highly homologous, with an identity of 66% and a similarity of 753 ⁇ 4.
  • Example 3 The gene encoding human autoimmune disease-related protein 16 was cloned by RT-PCR method. Total fetal brain cell RNA was used as a template, and ol igo-dT was used as a primer to perform reverse transcription reaction to synthesize CDM. PCR amplification with the following primers:
  • Primerl 5'-GGGCTTGGGTAAAAATGGCTGAAT-3 '(SEQ ID NO: 3)
  • Pr imer2 5'-AGTGAAACACGCAGCTTTATTAAG-3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp at the 5 'end of SEQ ID NO: 1;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification reaction conditions reaction volume containing 50 ⁇ 1 of 50ramol / LC 1, 10mmol / L Tri s- Cl, (pH8 5.), 1. 5mmol / L MgCl 2, 200 ⁇ mol / L dNTP, l Opmol primer, 1U Taq DNA polymerase (C 1 on tech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perk ynEmer) for 25 cycles under the following conditions: 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) using a TA cloning kit. D-sequence analysis showed that PCR The DM sequence of the product is exactly the same as that of 1-1174bp shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of human autoimmune disease-related protein 16 gene expression Total RNA was extracted in one step [Ana l. Biochera 1987, 162, 156-159].
  • This method involves acid guanidinium thiocyanate-chloroform extraction
  • the tissue is homogenized with 4M guanidinium 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), centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. Wash the obtained RNA precipitate with 70% ethanol, dry and dissolve in water.
  • RNA was electrophoresed on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-ImM EDTA-2.2 M formaldehyde. Then Transfer to a nitrocellulose membrane. Ct- 32 P dATP was used to prepare a labeled DNA probe by random primers.
  • the D probe used was the PCR amplified human autoimmune disease-related protein 16 coding region sequence shown in Figure 1 (682bp to 1113bp).
  • the 32P- labeled probes (about 2 ⁇ 10 6 cpm / ml) and RNA was transferred to nitrocellulose Membrane hybridized overnight at 42 ° C in a solution, the solution comprising 50% formamide -25m KH (pH7. 4) -5 x SSC- 5 x Denhardt, s solution, and 200 g / ml salmon sperm DNA 2 P0 4.
  • the filter was washed in 1 x SSC-0. 1% SDS for 30 min at 55 ° C. Then, it was analyzed and quantified by Phosphor Imager.
  • Example 5 In vitro expression and isolation of recombinant human autoimmune disease-related protein 16 And purification According to the sequence of the coding region shown in SEQ ID NO: 1 and Figure 1, a pair of specific amplification primers was designed, the sequence is as follows:
  • Pr imer3 5'-CCCCATATGATGAATGTGTGCGACAACCTTGGGG-3 '(Seq ID No: 5)
  • Pr imer4 5'-CCCGGATCCTTACATTATGGAGCCCGGGAGCCTG-3' (Seq ID No: 6)
  • the Ndel and BamHI restriction sites correspond to the expression vector plasmid pET-28b (+) (Novagen, Ca. No. 69865. 3) Selective endonuclease site.
  • PCR was performed using the PBS-0480G04 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-0480G04 plasmid, primers Primer-3 and Primer-4 were 1 Opmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1, respectively. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles. Ndel and BaraHI were used to double-digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligated product was transformed into coliform bacteria DH5 ct by the calcium chloride method. (Final concentration 30 ⁇ 8 / ⁇ 1) LB plates after overnight culture, kanamycin-containing screening by colony PCR, positive clones and sequenced. A positive clone (PET-0480G04) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (N 0va gen) using the calcium chloride method.
  • the host strain BL21 (pET-0480G04) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1 mmol / 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 6. Bind Quick Cartr idge (product of Novagen) was used. By chromatography, a purified human protein 16 related to the target protein was obtained.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine humaine 16 associée aux maladies auto-immunes, et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la protéine humaine 16 associée aux maladies auto-immunes.
PCT/CN2000/000405 1999-10-29 2000-10-30 Nouveau polypeptide, proteine humaine 16 associee aux maladies auto-immunes, et polynucleotide codant pour ce polypeptide WO2001032698A1 (fr)

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AU12660/01A AU1266001A (en) 1999-10-29 2000-10-30 A novel polypeptide-human autoimmune disease associated protein 16 and the polynucleotide encoding said polypeptide

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CN99119923.5 1999-10-29
CN 99119923 CN1302885A (zh) 1999-10-29 1999-10-29 一种新的多肽——人自身免疫疾病相关蛋白16和编码这种多肽的多核苷酸

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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHANG M. ET AL.: "Cloning and intracellular localization of the U2 small nuclear ribonucleoprotein auxiliary factor small subunit", PROC. NATL. ACAD. SCI. USA, vol. 89, no. 18, September 1992 (1992-09-01), pages 8769 - 8773 *

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