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WO2001046238A1 - Nouveau polypeptide, alpha-1b-glycoproteine humaine 12, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, alpha-1b-glycoproteine humaine 12, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001046238A1
WO2001046238A1 PCT/CN2000/000587 CN0000587W WO0146238A1 WO 2001046238 A1 WO2001046238 A1 WO 2001046238A1 CN 0000587 W CN0000587 W CN 0000587W WO 0146238 A1 WO0146238 A1 WO 0146238A1
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Prior art keywords
polypeptide
polynucleotide
glycoprotein
human alpha
sequence
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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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Shanghai Biowindow Gene Development Inc
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Priority to AU19855/01A priority Critical patent/AU1985501A/en
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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
    • C07K14/473—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used alpha-Glycoproteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, namely human alpha-IB-glycoprotein 12, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and the polypeptide. Background technique
  • Carbohydrates, proteins, and lipids are the main components of cell membranes. Among them, transmembrane glycoproteins catalyze reactions, transfer between molecules inside and outside the membrane, receive and transmit intracellular signals, membrane localization of enzyme substrates, tissue identification marks, etc Plays an important role.
  • Glycoproteins are antigens that can be obtained from many viral reactions. Glycoproteins and their antibodies can play a role in the diagnosis and treatment of diseases caused by many viruses, such as human acquired immune deficiency syndrome (AIDS).
  • HIV human immunodeficiency virus
  • the outer membrane glycoprotein is an important influencing factor both in the process of virus invasion and the cytopathic effects induced by it, but the specific mechanism is still unclear.
  • Alpha-IB-glycoprotein A1BG is a protoplasmic protein whose function has not been determined. It was discovered in 1986. (Proc. Nat. Acad. Sci. 83: 2363- 2367, 1986) A human A1B (; protein with an approximate molecular weight of 63KD) has been discovered. It consists of a polypeptide chain, the N-terminus of this polypeptide chain is connected to four Glucosamine oligosaccharides. This polypeptide chain has 5 disulfide bond structures within the chain and contains 474 amino acid residues. A1BG consists of 5 repeating functional domains, each of which contains about 95 Amino acid residues and a disulfide bond structure.
  • the polypeptide of the present invention was inferred and identified as a new human alpha-IB-glycoprotein 12 (HA1BG12), and its homologous protein was a human alpha-IB-glycoprotein that has been discovered.
  • HA1BG12 human alpha-IB-glycoprotein 12
  • its homologous protein was a human alpha-IB-glycoprotein that has been discovered.
  • research on the structure and function of the polypeptide of the present invention needs further development.
  • the human alpha-IB-glycoprotein 12 protein plays an important role in regulating important functions of the body such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, so there has been a need to identify more participation in the field These processes identify the human alpha-IB-glycoprotein 12 protein, especially the amino acid sequence of this protein.
  • the isolation of newcomer alpha-IB-glycoprotein 12 protein-encoding genes also provides the basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding DNA. Disclosure of invention
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding human alpha-IB-glycoprotein 12.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding human alpha-IB-glycoprotein 12.
  • Another object of the present invention is to provide a method for producing human alpha-1B-glycoprotein 12.
  • Another object of the present invention is to provide an antibody against a human alpha-IB-glycoprotein 12 of the polypeptide of the present invention.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors of human alpha-IB-glycoprotein 12 against the polypeptide of the present invention.
  • Another object of the present invention is to provide diagnostic treatment related to human alpha-IB-glycoprotein 12 abnormalities Of disease.
  • the present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 102 to 440 in SEQ ID NO: 1; and (b) a sequence having 1-498 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 alpha-1B-glycoprotein 12 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 in vitro detection of a disease or disease susceptibility associated with abnormal expression of human alpha-IB-glycoprotein 12 protein, which comprises detecting mutations in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, Alternatively, the amount or biological activity of a polypeptide of the invention in a biological sample is detected.
  • 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 polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of human alpha-IB-glycoprotein 12.
  • 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 refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response in appropriate animals or cells and to bind to specific antibodies.
  • An "agonist” refers to a molecule that, when combined with human alpha-IB-glycoprotein 12, can cause the protein to change, thereby regulating the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that binds human alpha-IB-glycoprotein 12.
  • Antagonist refers to a molecule that can block or modulate the biological or immunological activity of human alpha-IB-glycoprotein 12 when combined with human alpha-1B-glycoprotein-12 .
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that can bind human alpha-1B-glycoprotein 12.
  • Regulation refers to a change in the function of human alpha 1-IB-glycoprotein 12, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological properties and functions of human alpha 1-B-glycoprotein 12. Or changes in immune properties.
  • substantially pure means substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated.
  • Those skilled in the art can purify human alpha-IB-glycoprotein 12 using standard protein purification techniques.
  • Substantially pure human alpha-IB-glycoprotein 12 produces a single main band on a non-reducing polyacrylamide gel.
  • the purity of human a lpha-1B-glycoprotein 12 peptide 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 conditions with reduced stringency allow non-specific binding, because conditions with reduced stringency require that the two sequences bind to each other as either specific or selective interactions.
  • Percent identity refers to the percentage of sequences that are the same or similar in a comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (La sergene sof tware package, DNASTAR, Inc., Mad Son Wis.). The MEGAL IGN program can compare two or more sequences according to different methods such as Clus ter method (Higgins, D. G. and P. M. Sharp (1988) Gene 73: 237-244). The Cluster method arranges the groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula:
  • nucleic acid sequences X 100 Number of residues in sequence A-number of spacer residues in sequence A-number of spacer residues in sequence B
  • percent identity between nucleic acid sequences can also be determined by the Cluster method or by methods known in the art such as Jo t un He in (He in J., (1990) Methods in emzumo l ogy 183: 625-645) .
  • Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitutions for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and? It can specifically bind to the epitope of human al-pha-IB-glycoprotein 12.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not part of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • the polynucleotide and peptide in the natural state in a living cell are not separated and purified, but the same polynucleotide or polypeptide is separated and purified if it is separated from other substances existing in the natural state. of.
  • isolated human al pha-1B-glycoprotein 12 means that human al pha-1 B-glycoprotein 12 is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated.
  • the skilled artisan can purify human alpha-IB-glycoprotein 12 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human a lpha-IB-glycoprotein 12 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human a lpha-IB-glycoprotein 12, 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 invention may be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells). 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 alpha-IB-glycoprotein 12.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the human alpha-IB-glycoprotein 12 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or ( ⁇ ⁇ )
  • Such a polypeptide in which the mature polypeptide is fused with another compound such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV).
  • a polypeptide in which an additional amino acid sequence is fused into the mature polypeptide Sequences (such as leader sequences or secretory sequences or sequences used to purify this polypeptide or protease sequences)
  • sequences such as leader sequences or secretory sequences or sequences used to purify this polypeptide or protease sequences
  • 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.
  • Polynucleotides of the invention are found from a CDM library of human fetal brain tissue. It contains a polynucleotide sequence of 598 bases in length and its open reading frame (102-440) encodes 112 amino acids. According to the amino acid sequence homology comparison, it was found that this peptide has 98% homology with a human al pha-1B glycoprotein that has been discovered.
  • the human a lpha-IB-glycoprotein 12 has one Similar structure and function of human a lpha-IB-glycoprotein.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • D can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a “degenerate variant” refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity, between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% F i co ll, 42 ° C, etc .; or (3) only between the two sequences Crosses occur 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, most preferably at least 100 nucleotides. More than nucleotides.
  • Nucleic acid fragments can also be used for nucleic acid amplification technology (E.g., PCR) to identify and / or isolate a polynucleotide encoding human alpha-IB-glycoprotein 12.
  • the polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • polynucleotide sequence encoding the human alpha-IB-glycoprotein 12 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) Isolation of double-stranded genomic DNA I) NA sequence; 2) Chemical synthesis of DNA sequence to obtain double-stranded DM of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (DDNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of human alpha-IB-glycoprotein 12 transcript levels; (4) ) Detecting protein products of gene expression through immunological techniques or 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 generally 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).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product of human alpha-IB-glycoprotein 12 gene expression.
  • a method for amplifying DNA / RNA using PCR technology (Saiki, et al. Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-cDNA 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 determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, the sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising 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 alpha-IB-glycoprotein 12 coding sequence, and the recombinant technology to produce the polypeptide of the present invention Methods.
  • a polynucleotide sequence encoding human alpha-IB-glycoprotein 12 can be inserted into a vector to form 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 well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human alpha-IB-glycoprotein 12 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 p promoters of E.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenovirus enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human alpha-IB-glycoprotein 12 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a genetically engineered host 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.
  • 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 DNA uptake can be in the exponential growth phase were harvested, treated with CaC l 2 method used in the step are 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. Wait.
  • polynucleotide sequence of the present invention can be used to express or produce recombinant human alpha-1B-glycoprotein 12 (Scence, 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, recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid
  • FIG. 1 is a comparison diagram of amino acid sequence homology between the inventor a pha-IB-glycoprotein 12 and a discovered human a-pha-IB-glycoprotein.
  • the upper sequence is human a pha-1B-glycoprotein 12
  • the lower sequence is a human a pha-IB-glycoprotein that has been found.
  • 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 alpha-1B-glycoprotein 12. 12kDa is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention
  • the sequence of the human alpha-1B-glycoprotein 12 of the present invention and the protein sequence encoded by the human alpha-1B-glycoprotein 12 of the present invention were used with the Blast program (Basiclocal Alignment search tool) [Altschul, SF et al. J. Mol. Biol. 1990; 215: 403-10 ], Perform homology search in databases such as Genbank, Swissport.
  • the gene most homologous to the human alpha-IB-glycoprotein 12 of the present invention is a known human alpha-IB-glycoprotein that has been discovered.
  • the results of protein homology are shown in Figure 1. The two are highly homologous, with 98% identity; 100% similarity.
  • Example 3 Cloning of a gene encoding human alpha-1B-glycoprotein 12 by RT-PCR
  • CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer. After purification of Qiagene's kit, PCR amplification was performed with the following primers:
  • Primer 1 5'- ACGGCTGCGAGAAGACGAAGCTTAG —3, (SEQ ID NO: 3)
  • Primer2 5'- ACCTCTCTTCACATTTATTAATTC —3, (SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp of the 5th end of SEQ ID NO: 1;
  • Primer 2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification conditions 50 mmol / L KC1, 10ramol / L Tris-CI, (pH8.5), 1.5mraol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol primers in a reaction volume of 50 ⁇ 1 1U of Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min.
  • ⁇ -act in was set as positive during RT-PCR. Controls and template blanks were negative controls.
  • This method involves acid guanidinium thiocyanate-chloroform extraction. That is, 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 ) And centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • RNA was prepared using cc- 32 P dATP by random primers.
  • the DNA probe used was the human alpha-1B-glycoprotein 12 coding region sequence (102bp to 440bp) amplified by PCR as shown in FIG.
  • a 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 (pH 7.4)-5 x SSC- 5 x Denhardt's solution and 200 ⁇ g / ml salmon sperm DNA. After hybridization, filter was placed in 1 x SSC-0.1% SDS at 55. C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 5 In vitro expression, isolation and purification of recombinant human alpha-1B-glycoprotein 12 According to SEQ ID NO: 1 and the coding region sequence shown in FIG. 1, a pair of specific amplification primers is designed, and the sequences are as follows:
  • Primer3 5,-CCCCATATGATGGACGGCTGCGAGAAGACGAAGC -3, (Seq ID No: 5)
  • Priraer4 5'- CCCGGATCCGGAGCTCCTGGTGGCAGAAAGCTGA -3, (Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and BamHI digestion sites, respectively, followed by the coding sequences of the 5' and 3 'ends of the target gene, respectively.
  • the Ndel and BamHI restriction sites correspond to the selective endonuclease sites on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3).
  • the pBS-0050d07 plasmid containing the full-length target gene was used as a template for the PCR reaction.
  • PCR reaction conditions are as follows: a total volume of 50 ⁇ 1 contains 10 pg of pBS- 0050d07 plasmid, primers? 1 "11116]: -3 and? 1 1116]: -4 points and another!] Is 1 ( ⁇ 11101, Advantage polymerase Mix
  • a peptide synthesizer (product of PE company) was used to synthesize the following human alpha-IB-glycoprotein 12-specific peptides: NH 2 -Met-Asp-Gly-Cys-Glu-Lys-Thr-Lys-Leu-Arg-Thr- Ala-Ala-Arg-Arg-COOH (SEQ ID NO: 7).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • Compound with 1 ⁇ 2 g of the above hemocyanin polypeptide And Freund's adjuvant were used to immunize rabbits.
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in various aspects.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is identified whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can also be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissues or Whether the expression in pathological tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern blotting,
  • Northern blotting and copying methods etc., all use the same steps to fix the polynucleotide sample to be tested on the filter and then hybridize. These same steps are: The sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding sites 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. After the hybridization step, the unhybridized probes are removed by a series of membrane washing steps.
  • higher-intensity washing conditions such as lower salt concentration and higher temperature
  • 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 high-intensity washing conditions, the hybridization specificity of the first type of probe with the sample is the strongest and is retained.
  • the preferred range of probe size is 18-50 nucleotides
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, then the primary probe should not be used;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutant sequence of the gene fragment of SEQ ID NO: 1 or its complementary fragment (41Nt):
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membrane nitrocellulose membrane
  • High-intensity washing film 1) Take out the hybridized sample membrane.
  • Gene chip or gene microarray is a new technology currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • a total of 1,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotides of the present invention. They were amplified by PCR (as described in Example_), and the amplified product was purified to a concentration of about 500ng / ul, and spotted with a Cartesian 7500 spotting instrument (purchased from Cartesian, USA) at On glass media, the distance between points is 280 ⁇ m. The spotted slides were hydrated, dried, and cross-linked in a UV cross-linking instrument. After elution, the DNA was fixed on the glass slides to prepare chips. The specific method steps have been variously reported in the literature. The post-spotting processing steps of this embodiment are:
  • Total mRNA was extracted from normal liver and liver cancer tissues by a single method, and the mRNA was purified using Oligotex mRNA Midi Kit (purchased from QiaGen).
  • the fluorescent reagent Cy3dUTP (5- Amino- propargy 1-2 ⁇ - deoxyur i dine 5'- triphate coupled to Cy3 fluorescent dye (purchased from Amer sham Phamac ia Biotech) was used to label the mRNA of normal liver tissue, and the fluorescent reagent Cy5dUTP (5-Amino-propargyl-2'-deoxyur idine 5 * -tr iphate coupled to C fluorescent dye (purchased from Araersham Phamac ia Biotech) was used to label the abnormal liver tissue mRNA, and the probe was prepared after purification.
  • the probes from the above two tissues and the chip were respectively hybridized in a UniHyb TM Hybrid Izat ion Solut ion (purchased from TeleChem) hybridization solution for 16 hours, and the washing solution (1 x SSC, 0.2% SDS) ) After washing, scan with a ScanArray 3000 scanner (purchased from Genera Scanning, USA). The scanned images are analyzed by Imagene software (Biodi scovery, USA), and the Cy3 / Cy5 ratio of each point is calculated. The ratio Points less than 0.5 and greater than 2 are considered genes with differential expression.
  • the polypeptide (human al-pha-IB-glycoprotein 12) of the present invention belongs to the human glycoprotein family, and it is presumed to catalyze the reaction, transfer the molecule inside and outside the membrane, receive and transmit intracellular signals, membrane localization of the enzyme substrate, Tissue recognition markers and other aspects play an important role.
  • the human alpha-1B-glycoprotein 12 of the present invention has homology with immunoglobulins, and it can be speculated that they are functionally related.
  • the human al-pha-IB-glycoprotein 12 of the present invention can be directly used as a drug or vaccine for the deduction and treatment of many diseases, such as human acquired immune deficiency syndrome (AIDS), malignant tumors, immune diseases, and endocrine system. Diseases, neurological diseases, etc.
  • AIDS human acquired immune deficiency syndrome
  • Various tumors include: including epithelial tissue (such as basal epithelium, squamous epithelium, mucus cells, etc.), (such as fibrous tissue, adipose tissue, cartilage tissue, smooth muscle tissue, vascular and lymphatic endothelial tissue, etc.), hematopoietic tissue ( (Such as B cells, T cells, tissue cells, etc.), central nervous tissue, peripheral nerve tissue, endocrine tissue, gonadal tissue, special tissue (such as dental tissue, etc.) derived tumors, such as gastric cancer, liver cancer, colorectal cancer, breast Cancer, lung cancer, prostate cancer, cervical cancer, pancreatic cancer, esophageal cancer, etc.
  • epithelial tissue such as basal epithelium, squamous epithelium, mucus cells, etc.
  • fibrous tissue such as fibrous tissue, adipose tissue, cartilage tissue, smooth muscle tissue, vascular and lymphatic endothelial tissue, etc.
  • the polypeptide of the present invention is an immunomodulator and has an immune promoting or immunosuppressing effect.
  • the polypeptide of the present invention can be used for the treatment of diseases including non-response of immune response, or abnormal immune response, or ineffective host defense. Damage, defect or damage to the immune tissue by the polypeptide of the present invention and its antibody Disorders also play a role, especially for hematopoietic diseases (such as malignant anemia), skin diseases (such as psoriasis), autoimmune diseases (such as rheumatoid arthritis), radiation diseases, and the generation and regulation of immune lymphocytes. Relationship.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human alpha-IB-glycoprotein 12.
  • Agonists enhance human alpha-IB-glycoprotein 12 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 membrane preparations expressing human alpha-1B-glycoprotein 12 can be cultured with labeled human alpha-IB-glycoprotein 12 in the presence of drugs. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human alpha-1B-glycoprotein 12 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human alpha-IB-glycoprotein 12 can bind to human alpha-IB-glycoprotein 12 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 function biological functions.
  • human alpha-1B-glycoprotein 12 When screening compounds as antagonists, human alpha-1B-glycoprotein 12 can be added to bioanalytical assays to determine compounds by measuring their effect on the interaction between human alpha-IB-glycoprotein 12 and its receptor Whether it is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same way as for screening compounds described above. Polypeptide molecules capable of binding to human alpha-1B-glycoprotein 12 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. In screening, human alpha-1B-glycoprotein 12 molecules should generally be labeled.
  • the present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies directed against human alpha-IB-glycoprotein 12 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 produced by injecting human alpha-IB-glycoprotein 12 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 Adjuvant, etc.
  • Techniques for preparing monoclonal antibodies to human alpha-IB-glycoprotein 12 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, Human B-cell hybridoma technology, EB-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions to 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 (t ;. S. Pat No. 4946778) can also be used to produce single chain antibodies against human alpha-IB-glycoprotein 12.
  • Anti-human alpha-IB-glycoprotein 12 antibodies can be used in immunohistochemistry to detect human alpha-IB-glycoprotein 12 in biopsy specimens. Monoclonal antibodies that bind to human alpha-IB-glycoprotein 12 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 alpha-1B-glycoprotein 12 high affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of the antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human alpha-IB-glycoprotein 12 positive Cell.
  • the antibodies of the present invention can be used to treat or prevent human alpha-IB-glycoprotein 12-related diseases. Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human alpha-IB-glycoprotein 12.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human alpha-IB-glycoprotein 12 levels. These tests are well known in the art and include FISH assays and radioimmunoassays.
  • the levels of human alpha-IB-glycoprotein 12 detected in the test can be used to explain the importance of human alpha-lB-glycoprotein 12 in various diseases and to be useful in the diagnosis of human alpha-1B-glycoprotein 12 Disease.
  • 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 alpha-1B-glycoprotein 12 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 alpha-IB-glycoprotein 12.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human alpha-IB-glycoprotein 12 to inhibit endogenous human alpha-IB-glycoprotein 12 activity.
  • a variant human alpha-IB-glycoprotein 12 may be shortened human alpha-IB-glycoprotein 12 lacking a signaling domain, and although it can bind to downstream substrates, it lacks signaling. Ductive activity.
  • the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of human alpha-1B-glycoprotein 12.
  • 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 alpha-IB-glycoprotein 12 into cells.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding human alpha-IB-glycoprotein 12 can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human alpha-IB-glycoprotein 12 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 alpha-1B-glycoprotein 12 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technology, such as solid-phase phosphoramidite chemical synthesis to synthesize oligonucleotides.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA. This DNA sequence has been integrated downstream of the vector's RNA polymerase promoter. In order to increase the stability of the nucleic acid molecule, 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 al pha-IB-glycoprotein 12 can be used for the diagnosis of diseases related to human al pha-1B-glycoprotein 12.
  • a polynucleotide encoding human al pha-1B-glycoprotein 12 can be used to detect the expression of human a lpha-IB-glycoprotein 12 or the abnormal expression of human al pha-IB-glycoprotein 12 in a disease state.
  • the DNA sequence encoding human al pha-1B-glycoprotein 12 can be used to hybridize biopsy specimens to determine the expression status of human alpha-1B-glycoprotein 12. Hybridization techniques include Southern blotting, Nor thern blotting, in situ hybridization, and the like.
  • polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissue.
  • Human al pha-IB-glycoprotein 12 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect human al pha-IB-glycoprotein 12 transcription Product.
  • Detection of mutations in the human alpha-IB-glycoprotein 12 gene can also be used to diagnose human alpha-1B-glycoprotein 12-related diseases.
  • Human alpha-IB-glycoprotein 12 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type human alpha-IB-glycoprotein 12 DNA sequences. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for marking chromosome positions.
  • the important first step is to locate these DNA sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared from the cDNA, and the sequences can be located on the 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 physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendelian Inheritance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions. Next, the differences in cDNA or genomic sequences between the affected and unaffected individuals need to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease.
  • Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable using cDNA sequence-based PCR.
  • the cDNA accurately mapped to the 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 alpha-1B-glycoprotein 12 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dose range of a l pha-IB-glycoprotein 12 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.

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Abstract

L'invention concerne un nouveau polypeptide, une alpha-1B-glycoprotéine humaine 12, 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 l'alpha-1B-glycoprotéine humaine 12.
PCT/CN2000/000587 1999-12-22 2000-12-18 Nouveau polypeptide, alpha-1b-glycoproteine humaine 12, et polynucleotide codant pour ce polypeptide Ceased WO2001046238A1 (fr)

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CN 99125693 CN1300756A (zh) 1999-12-22 1999-12-22 一种新的多肽－人alpha－1B－糖蛋白12和编码这种多肽的多核苷酸
CN99125693.X 1999-12-22

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CN113092771A (zh) * 2019-12-23 2021-07-09 首都医科大学附属北京世纪坛医院 尿液α-1B-糖蛋白及其多肽片段在过敏性疾病中的应用

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DATABASE GENBANK [online] 10 September 1999 (1999-09-10), Database accession no. GI:69990 *
PROC. NATL. ACAD. SCI. USA, vol. 83, no. 8, 1986, pages 2363 - 2367 *

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