WO2001040299A1 - Nouveau polypeptide, prolipoproteine humaine 14 peptide signal, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, prolipoproteine humaine 14 peptide signal, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001040299A1
WO2001040299A1 PCT/CN2000/000514 CN0000514W WO0140299A1 WO 2001040299 A1 WO2001040299 A1 WO 2001040299A1 CN 0000514 W CN0000514 W CN 0000514W WO 0140299 A1 WO0140299 A1 WO 0140299A1
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Prior art keywords
polypeptide
polynucleotide
signal peptide
precursor protein
protein signal
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PCT/CN2000/000514
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Bioroad Gene Development Ltd. Shanghai
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Priority to AU16895/01A priority Critical patent/AU1689501A/en
Publication of WO2001040299A1 publication Critical patent/WO2001040299A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, a human lipoprotein precursor protein signal peptide 14, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique
  • Lipoproteins are a class of binding proteins composed of proteins and various fatty acids and steroids. Proteins and steroids are bound together by weak non-covalent bonds, while other lipoproteins are bound by proteins and lipids by covalent bonds. Lipoproteins can be divided into three categories based on their protein composition, nuclear proteins ( (Such as thrombin to live enzymes), phosphoproteins (such as lipophosphoproteins in yolk), and simple proteins (such as plasma lipoproteins and brain proteolipids). The role of lipoproteins in the body is very important. For example, part of the lipids in the blood is transmitted by plasma lipoproteins
  • Covalent modification of lipids to membrane proteins is a common phenomenon in cells.
  • the proteins at the cell membrane are all lipoproteins. They interact with the phospholipid layer of the cell membrane to form a membrane structure.
  • membrane lipoprotein synthesis and modification are more clearly studied in prokaryotes.
  • membrane lipoproteins are first synthesized as a protein precursor.
  • the N-terminus of this precursor protein contains a signal peptide.
  • two processes of modification of the N-terminal cysteine and removal of the signal peptide occur successively. This process takes place in a conservative area.
  • the signal peptide of a protein that has not undergone lipid modification will not have this conserved sequence. It appears that this conserved sequence is a sorting signal for whether the mature protein is lipid-modified or not.
  • signal peptidase II recognizes a conserved sequence on the signal peptide and cleaves the upstream segment of the cysteine residue connected to the glycerolipid-fatty acid lipid. This conserved sequence is:
  • apolipoprotein is associated with Alzheimer's disease.
  • High-density lipoprotein can remove excess cholesterol present in the brain and keep the starch mass at a normal level. Abnormal expression of this type of lipoprotein causes Alzheimer's disease, cholesterol deposits in the brain, and brain tumors.
  • liver diseases such as exogenous / endogenous lipidemia, familial hypercholesterolemia, cholesterol storage disease, fatty liver, etc. Severe fatty liver can also cause liver cancer. Lipoprotein anabolic disorders can also cause diseases such as coronary heart disease.
  • the polypeptide of the present invention contains a lipid-binding site of a membrane lipoprotein precursor signal peptide, and analysis of the expression profile indicates that the content of sub-peptide in liver cancer cells is lower than that in normal liver tissue, indicating that it is related to liver cancer and membrane lipids.
  • the function of the protein precursor signal peptide is consistent.
  • the present invention is a novel human lipoprotein precursor protein signal peptide, which has similar biological functions. And named as human lipoprotein precursor protein signal peptide 14.
  • the human lipoprotein precursor protein signal peptide 14 protein plays an important role in important body functions as described above, and it is believed that a large number of proteins are involved in these regulatory processes, there has been a need in the art to identify more human lipoproteins involved in these processes.
  • Precursor protein signal peptide 14 protein especially the amino acid sequence of this protein is identified. Isolation of the new human lipoprotein precursor protein signal peptide 14 protein encoding gene also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for developing diagnostic and / or therapeutic drugs, so isolating its coding DNA is important. It is an object of the present invention to provide isolated novel polypeptides-human lipoprotein precursor protein signal peptide 14 and fragments, analogs and derivatives thereof.
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a human lipoprotein precursor protein signal peptide 14.
  • Another object of the present invention is to provide a method for producing a human lipoprotein precursor protein signal peptide 14.
  • Another object of the present invention is to provide an antibody against the polypeptide-human lipoprotein precursor protein signal peptide 14 of the present invention.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention-human lipoprotein precursor protein signal peptide 14.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of the human lipoprotein precursor protein signal peptide 14.
  • the present invention relates to an isolated polypeptide.
  • the polypeptide is of human origin and comprises: SEQ ID No. 2 Amino acid sequence of a polypeptide, or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 619-1038 in SEQ ID NO: 1; and (b) having a sequence of 1-2572 in SEQ ID NO: 1 Sequence of bits.
  • the invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • a vector in particular an expression vector, containing the polynucleotide of the invention
  • a host cell genetically engineered with the vector including a transformed, transduced or transfected host cell
  • a method comprising culturing said Host cell and method of preparing the polypeptide of the 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 lipoprotein precursor protein signal peptide 14 protein, which comprises utilizing the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for in vitro detection of a disease or susceptibility to disease associated with abnormal expression of a human lipoprotein precursor protein signal peptide 14 protein, comprising 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 lipoprotein precursor protein signal peptide 14.
  • 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
  • this "multiple "Peptide” or “protein” is not meant to limit the amino acid sequence to the complete natural amino acid associated with the protein molecule.
  • a protein or polynucleotide “variant” 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 substituted amino acid has a structural or chemical property similar to the original amino acid, such as the replacement of 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 proteins with the structural, regulatory, or biochemical functions of natural molecules.
  • immunologically active refers to natural, recombinant, or synthetic proteins and fragments thereof in suitable animals or cells The ability to induce specific immune responses and to bind specific antibodies.
  • An "agonist” refers to a molecule that, when combined with a human lipoprotein precursor protein signal peptide 14, can cause the protein to change, thereby regulating the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind the human lipoprotein precursor protein signal peptide 14.
  • Antagonist refers to a molecule that can block or regulate the biological or immunological activity of human lipoprotein precursor protein signal peptide 14 when combined with human lipoprotein precursor protein signal peptide 14.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that can bind to human lipoprotein precursor protein signal peptide 14.
  • “Regulation” refers to a change in the function of human lipoprotein precursor protein signal peptide 14, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological properties and functions of human lipoprotein precursor protein signal peptide 14. Or changes in immune properties.
  • substantially pure ' means substantially free of other proteins, lipids, carbohydrates or other substances naturally associated with it.
  • Those skilled in the art can purify the human lipoprotein precursor protein signal peptide 14 using standard protein purification techniques, _, Basically pure human lipoprotein precursor protein signal peptide 14 can produce a single main band of human lipoprotein precursor protein signal peptide 14 on a non-reducing polyacrylamide gel. The purity of the polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to polynucleotides that naturally bind through base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence "CT-G-A” can be combined with the complementary sequence "G-A-CT”.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands The efficiency and intensity of hybridization have a significant effect.
  • “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 blotting or Nor thern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other specifically or selectively.
  • Percent identity refers to the percentage of sequences that are the same or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as through the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Cluster method (Higgins, D. G. and P. M. Sharp (1988) Gene 73: 237-244). The C l uster method arranges each group of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: The number of matching residues between sequence A and sequence B
  • the percent identity between nucleic acid sequences can also be determined by the Cluster method or by methods known in the art such as Jotun He in (He in J., (1990) Methods in emzumo logy 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 the “sense strand”.
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. Such a chemical modification may be the replacement of a hydrogen atom with an alkyl group, an acyl group or an amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological characteristics of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and? Which can specifically bind The epitope of human lipoprotein precursor protein signal peptide 14.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it occurs naturally).
  • a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system.
  • Such a polynucleotide may be part of a vector, or such a polynucleotide or polypeptide may be part of a composition. Since the carrier or composition is not part of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state. .
  • isolated human lipoprotein precursor protein signal peptide 14 means that human lipoprotein precursor protein signal peptide 14 is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify the human lipoprotein precursor protein signal peptide 14 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human lipoprotein precursor protein signal peptide 14 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, a human lipoprotein precursor protein signal peptide 14, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques.
  • polypeptide of the invention may be glycosylated, or it may be non-glycosylated.
  • the polypeptides of the invention may also include or exclude the initial methionine residue.
  • the present invention also includes fragments, derivatives, and analogs of the human lipoprotein precursor protein signal peptide 14.
  • fragment means substantially maintaining the present invention Human lipoprotein precursor protein signal peptide 14 A polypeptide with the same biological function or activity.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution The amino acid may or may not be encoded by the genetic codon; or ( ⁇ ) a type in which one or more amino acid residues A certain group is substituted by another group to include a substituent; or (III) is one in which the fused polypeptide is fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) A polypeptide sequence (such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protease sequence) formed by fusing additional amino acid sequences into a mature polypeptide.
  • a polypeptide sequence such as a leader sequence or a secreted sequence or a sequence used to purify this polypeptide or a protease
  • 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 with a total length of 2572 bases, and its open reading frame (6191038) encodes 139 amino acids.
  • This polypeptide has a characteristic sequence of a membrane lipoprotein lipid binding site, and it can be deduced that the human lipoprotein precursor protein signal peptide 14 has a structure and function represented by a membrane lipoprotein lipid binding site.
  • 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 it may be 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 that includes the polypeptide and a polynucleotide that includes 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.
  • This polynucleotide variant can be a naturally occurring allelic variant or a non-naturally occurring variant.
  • These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity between the two sequences).
  • the invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 6 (TC; 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 Between Hybridization occurs only when the identity is at least 95%, and more preferably 97%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nuclei. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding human lipoprotein precursor protein signal peptide 14.
  • 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 lipoprotein precursor protein signal peptide 14 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 DM fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DM sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • Various methods have been developed for mRNA extraction, and kits are also commercially available (Q i agene).
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Moleculiar Cling, A Labora tory Manual, Coldspring Harbor Laboraty. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Cloieitech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DM or DM-RNA hybridization; (2) the appearance or loss of marker gene function; (3) determination of the level of transcript of human lipoprotein precursor protein signal peptide 14 (4) Detecting protein products expressed by genes 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. In addition, the length of the probe is usually within 2,000 nucleotides, preferably within 1 000 nucleotides.
  • the probe used here is generally a DM sequence chemically synthesized based on the gene sequence information of the present invention. The genes or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase). In the (4) method, immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product of the human lipoprotein precursor protein signal peptide 14 gene expression.
  • a method of applying a PCR technique to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide 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 the polynucleotide of the present invention, and a host cell that is genetically engineered using the vector of the present invention or directly using the human lipoprotein precursor protein signal peptide 14 coding sequence, and that recombinant technology is used to produce the present invention.
  • Polypeptide method comprising the polynucleotide of the present invention, and a host cell that is genetically engineered using the vector of the present invention or directly using the human lipoprotein precursor protein signal peptide 14 coding sequence, and that recombinant technology is used to produce the present invention. Polypeptide method.
  • a polynucleotide sequence encoding the human lipoprotein precursor protein signal peptide 14 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors 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.
  • PL promoter eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, retroviral LTRs and other known controllable genes in prokaryotic cells or Promoters expressed in eukaryotic cells or their viruses.
  • 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. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, tumorigenic enhancers on the late side of the origin of replication, and adenoviral enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • the polynucleotide encoding the human lipoprotein precursor protein signal peptide 14 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.
  • Transforming a host cell with the DNA sequence of the present invention or a recombinant vector containing the DNA sequence can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaC I.
  • the steps used 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.
  • the polynucleotide sequence of the present invention can be used to express or produce a recombinant human lipoprotein precursor protein signal peptide 14 (Scence, 1984; 224: 1431). Generally, the following steps are taken:
  • step (3) Isolate and purify protein from culture medium or cells.
  • 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.
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be separated 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 chromatography
  • Figure 1 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated human lipoprotein precursor protein signal peptide 14. 14kDa is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention
  • poly (A) mRNA is reverse transcribed to form cDNA.
  • a Smart cDNA cloning kit (purchased from Clontech) was used to orient the cDNA fragment into the multiple cloning site of the pBSK (+.) Vector (Clontech) to transform DH5a. The bacteria formed a cDNA library.
  • Dye terminate cycle reaction ion sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer) were used to determine the sequences at the 5 'and 3' ends of all clones.
  • the determined cDNA sequence was compared with the existing public DM sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0558e09 was new DNA. Insert a cDNA fragment into the clone by synthesizing a series of primers Segments are measured in both directions. The results showed that the 0558e09 clone contained a full-length cDNA of 2572bp (as shown in Seq IDN0: 1), and a 139bp open reading frame (0RF) from 619bp to 1038bp, encoding a new protein (such as Seq ID NO: 2). We named this clone pBS-0558e09 and the encoded protein was named human lipoprotein precursor protein signal peptide 14.
  • Example 2 Cloning of a gene encoding human lipoprotein precursor protein signal peptide 14 by RT-PCR
  • CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer.
  • PCR amplification was performed with the following primers:
  • Primer 1 5'_GGAAAAATTTCTGTACATTTTCTCAA- 3 '(SEQ ID NO: 3)
  • Primer2 5,-GAGGAAACATAATCTGTATTTCAGTT- 3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp at the 5 'end of SEQ ID NO: 1;
  • Primer 2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification reaction conditions 50 ⁇ l of KC1, 10 mmol / L Tris-CI, (pH 8.5), 1.5 mmol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol primers in a 50 ⁇ 1 reaction volume , 1U 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 a positive control and template blank was set as a negative control.
  • the amplified product was purified using a QIAGEN kit, and ligated to a pCR vector (Invitrogen product) using a TA cloning kit.
  • the DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1-2572bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human lipoprotein precursor protein signal peptide 14 gene expression:
  • This method involves acid guanidinium thiocyanate-chloroform extraction. I.e. with 4M guanidinium isothiocyanate -25mM sodium citrate, 0.2M sodium acetate (P H4.0) of the tissue was homogenized phenol, 1 volume and 1/5 volume of chloroform - isoamyl alcohol (49: 1) Centrifuge after mixing. The aqueous layer was aspirated, isopropanol (0.8 vol) was added and the mixture was centrifuged to obtain RM precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • RNA probes were the human lipoprotein precursor protein signal peptide 14 coding region sequence (619bp to 1038bp) amplified by PCR.
  • a 32P-labeled probe (approximately 2 x 10 6 cpm / ml) and an RNA-transferred nitrocellulose membrane were placed in a solution at 42 ° C. C hybridization overnight, this solution contains 50. /. Formamide-25mM KH 2 P0 4 (pH7.4) -5 xSSC- 5 xDenhardt's solution and 20 ( ⁇ g / ml salmon sperm DNA. After hybridization, the filter was set at 1 ⁇ SSC- 0.1. /. Wash in SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 4 In vitro expression, isolation and purification of recombinant human lipoprotein precursor protein signal peptide 14
  • Primer 3 5'- CCCCATATGATAGCTATCATGTGTTTGAATCAGA- 3 '(Seq ID No: 5)
  • Primer4 5'- CCCGGATCCTCAGCATTCACAAAGGCAGCCAACC- 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 ′ end and the end of the target gene, respectively.
  • the Ndel and BamHI digestion sites correspond to the expression vector Selective endonuclease site on plasmid pET-28b (+) (Novagen, Cat. No. 69865.3).
  • PCR was performed using the pBS-0558e09 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 P BS_0558e09 plasmid, primers Primer-3 and Primer- 4 points, and 1 J for lpmol, Advantage polymerase Mix
  • Cycle parameters 94 ° C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles.
  • Ndel and BamHI were used to double-digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligation product was transformed into Ca. bacillus DH5a by the calcium chloride method.
  • NH 2 -Met-Cys-Leu-Asn-Gln-Arg-Phe-Glu-Leu-Tyr-Tyr-Leu-Glu-Asn-Asp- C00H (SEQ ID NO: 7).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin For methods, see: Avrameas, et al. Iramunochemistry, 1969; 6: 43. With 4mg of hemocyanin Peptide complex plus complete Freund's adjuvant was used to immunize rabbits. After 15 days, hemocyanin peptide complex plus incomplete Freund's adjuvant was used to boost immunity once.
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is identified whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can also be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissues or Whether the expression in pathological tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern imprinting, Nor thern blotting, and copying methods. They all use the same steps to fix the polynucleotide sample to be tested on the filter and then hybridize.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid.
  • unhybridized probes are removed by a series of membrane washes.
  • This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • oligonucleotide fragments for use as hybridization probes from the polynucleotide SEQ ID NO: 1 of the present invention should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • 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, the primary probe should not be used;
  • Probe 1 (probel), which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt)
  • Probe 2 which belongs to the second type of probe, is equivalent to the replacement mutation sequence (41Nt) of the gene fragment of SEQ ID NO: 1 or its complementary fragment:
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • Two NC membranes are required for each probe, so that it can be used in the following experimental steps.
  • the film was washed with high-strength conditions and strength conditions, respectively.
  • probes 1 3 ⁇ IProbe (0.1 OD / 10 ⁇ 1), add 2 ⁇ IKinase buffer, 8-10 uCi ⁇ - 32 P- dATP + 2U Kinase, to make up to a final volume of 20 ⁇ 1.
  • the 32 P-Probe (the second peak is free ⁇ - 32P -dATP) is prepared.
  • pre-hybridization solution 10xDenhardt's; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA).
  • Gene microarrays or DNA microarrays are new technologies currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of large numbers 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 .
  • the specific method steps have been reported in the literature. For example, see DeRisi, JL, Lyer, V. & Brown, P.0. (1997) Science 278, 680-686. And Helle, RA, Schema, M. , Chai, A., Shalom, D., (1997) PNAS 94: 2150-2155.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were respectively amplified by PCR. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and spotted on a glass medium using a Cartesian 7500 spotter (purchased from Cartesian, USA). The distance is 280 ⁇ . The spotted slides were hydrated, dried, and cross-linked in a purple diplomatic coupling instrument. After elution, the DNA was fixed on a glass slide to prepare a chip.
  • the specific method steps have been reported in the literature in various ways, and the specific method steps have been reported in the literature in various ways.
  • the spotting post-processing steps of this embodiment are:
  • Total mRNA was extracted from normal liver and liver cancer by one-stroke method, and Oligotex mRNA Midi Kit (purchased from QiaGen) was used to purify mRNA, and the fluorescent reagent Cy3dUTP (5-Amino- propargy 1 ⁇ 2 ⁇ -deoxyur i dine 5 '-tr iphate coupled to Cy3 fluorescent dye was purchased from Amersham Phamacia Biotech Company by reverse transcription. ) Mark normal tissue mRNA. Mark liver cancer tissue mRNA with Cy5dUTP (5-Amino-propargyl-2 '-deoxyur i dine 5 * -tr iphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech).
  • Probes were prepared after purification. Specific steps refer to and methods Schena, M., Shalon, D., Heller, R. (1996) Proc. Natl. Acad. Sci. USA. Vol. 93: 10614- 10619. Schena, M., Shalon, Dar i. Davis, RW (1995) Science.270. (20): 467-480. (3) Hybridization
  • Probes from the above two tissues and chips were hybridized in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, washed with a washing solution (1 x SSC, 0.2% SDS) at room temperature, and then scanned with ScanArray 3000.
  • Scanner purchased from General Scanning Company, USA
  • the scanned image is processed by Imagene software (Biodiscovery Company, USA) for data analysis, and the Cy3 / Cy5 ratio of each point is calculated.
  • the points whose ratio is less than 0.5 and greater than 2 are considered Genes with differential expression.
  • polypeptides of the present invention as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.
  • this signal peptide sequence can treat or prevent diseases caused by a lack of normal functioning lipoproteins due to abnormal signal peptide synthesis, such as Alzheimer's disease, brain tumors, exogenous / endogenous lipidemia, family Hypercholesterolemia, Cholesterol lipid storage disease, fatty liver, liver cancer and coronary heart disease.
  • the polypeptides or fragments thereof of the present invention can also be used to treat or prevent various cancers, including but not limited to: Respiratory system tumors: nasal and sinus tumors, nasopharyngeal cancer, laryngeal cancer, tracheal tumors, lung cancer, pleural mesothelioma digestive system
  • Tumors salivary gland tumor, esophageal cancer, esophageal leiomyosarcoma, primary esophageal small cell cancer, gastric cancer, gastric malignant lymphoma, gastric carcinoid, colorectal cancer, colon cancer, intestinal malignant lymphoma, primary liver cancer, liver mother Cell tumor, primary gallbladder cancer, pancreatic cancer
  • lymphatic tumors acute leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, malignant lymphoma (such as lymphatic reticulum, malignant lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, etc.), malignant Histiocytosis Neurological tumors: astrocytoma, ependymal tumor, medulloblastoma, meningiomas, glioblastoma, acoustic neuroma, angiogenic tumor, pituitary adenoma, craniopharyngioma
  • osteoid osteoma osteochondroma, chondroma, osteoblastoma, chondroblastoma, etc.
  • malignant bone tumors such as giant cell tumor of bone, osteosarcoma, chondrosarcoma, Ewing's sarcoma, myeloma
  • Tumors of the genitourinary system benign tumors such as renal tubular adenoma, eosinophilic adenoma, juxtaglomerular cell tumor, polycystic kidney tumor, seminoma, teratoma, testicular stromal cell tumor, intrauterine Membrane stromal tumor, hydatidiform mole, ovarian tumor, breast fibroma, malignant tumors such as renal cell carcinoma, renal sarcomatoid carcinoma, papillary renal cell carcinoma, nephroblastoma, prostate cancer, testicular tumor chorionic carcinoma, cervical cancer , Endometrial cancer, endometrial cancer, infusion tube cancer, ovarian cancer, breast cancer
  • benign tumors such as renal tubular adenoma, eosinophilic adenoma, juxtaglomerular cell tumor, polycystic kidney tumor, seminoma, teratoma, testicular stromal cell tumor, intrauterine Membr
  • Endocrine system tumors pituitary adenoma, benign thyroid tumor, thyroid cancer, parathyroid adenoma, parathyroid cancer, adrenal myeloma, pheochromocytoma, islet cell tumor, multiple endocrine gland tumor, thymic tumor
  • Soft tissue tumors fibroma, fibrosarcoma, fibromatosis, lipoma, liposarcoma, leiomyoma, leiomyosarcoma, rhabdomyosarcoma, rhabdomyosarcoma, synovial tissue tumor, hemangioma, intramuscular hemangioma, blood vessels Globuloma, hemangioendothelial sarcoma, lymphangioma, lymphangiomyoma, lymphatic endothelial sarcoma, histiocytoma, malignant fibrous histiocytoma, soft tissue acinar sarcoma, clear cell sarcoma, myxoma, extraosteal Ewing sarcoma, Soft tissue osteosarcoma, soft tissue chondrosarcoma, mesothelioma, epithelioid sarcoma, schwannomas, neurofibromas, malignant schw
  • Skin malignancies dermal Mike cell tumor, Kaposi sarcoma, melanoma
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) the human lipoprotein precursor protein signal peptide 14.
  • Agonists enhance human lipoprotein precursor protein signal peptide 14 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 lipoprotein precursor protein signal peptide 14 can be cultured with labeled human lipoprotein precursor protein signal peptide 14 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human lipoprotein precursor protein signal peptide 14 include antibodies, compounds, receptor deletions, and the like that have been screened.
  • the antagonist of human lipoprotein precursor protein signal peptide 14 can bind to human lipoprotein precursor protein signal peptide 14 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide to make the polypeptide Cannot perform biological functions.
  • human lipoprotein precursor protein signal peptide 14 can be added to the bioanalytical assay by determining the effect of the compound on the interaction between human lipoprotein precursor protein signal peptide 14 and its receptor. Determine if the compound is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to human lipoprotein precursor protein signal peptide 1 4 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, the human lipoprotein precursor protein signal peptide 14 molecule should generally be labeled.
  • the present invention provides polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. To produce antibodies. These antibodies can be polyclonal or monoclonal antibodies. The invention also provides antibodies against the human lipoprotein precursor protein signal peptide 14 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human lipoprotein precursor protein signal peptide 14 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to 'S adjuvant and so on.
  • Techniques for preparing monoclonal antibodies to human lipoprotein precursor protein signal peptide 14 include, but are not limited to, hybridoma technology (Kohler and Milste in. Nature, 1975, 256: 495-497), triple tumor technology, human beta -Cell hybridoma technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions and non-human variable regions can be produced using existing techniques (Morri et al, PNAS, 1985, 81: 6851). 0 Existing techniques for producing single-chain antibodies (US Pa t No. 4946778) can also be used to produce single chain antibodies against human lipoprotein precursor protein signal peptide 14.
  • Antibodies against human lipoprotein precursor protein signal peptide 14 can be used in immunohistochemical techniques to detect human lipoprotein precursor protein signal peptide 14 in biopsy specimens.
  • Monoclonal antibodies that bind to human lipoprotein precursor protein signal peptide 14 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.
  • 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 the human lipoprotein precursor protein signal peptide 14 Positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to the human lipoprotein precursor protein signal peptide 14. Administration of an appropriate dose of antibody can stimulate or block the production or activity of human lipoprotein precursor protein signal peptide 14.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human lipoprotein precursor protein signal peptide 14 levels.
  • These tests are well known in the art and include human lipoprotein precursor protein signal peptide 14 levels detected in FI SH assays and radioimmunoassay assays, and can be used to explain human lipoprotein precursor protein signal peptide 14 levels in various Importance in disease and in the diagnosis of diseases where human lipoprotein precursor protein signal peptide 14 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. Analysis.
  • the polynucleotide encoding human lipoprotein precursor protein signal peptide 14 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 lipoprotein precursor protein signal peptide 14.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human lipoprotein precursor protein signal peptide 14 to inhibit endogenous human lipoprotein precursor protein signal peptide 14 activity.
  • a variant human lipoprotein precursor protein signal peptide 14 may be a shortened human lipoprotein precursor protein signal peptide 14 that lacks a signaling functional domain. Although it can bind to downstream substrates, it lacks signal transduction. active.
  • the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human lipoprotein precursor protein signal peptide 14.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding a human lipoprotein precursor protein signal peptide 14 into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a human lipoprotein precursor protein signal peptide 14 can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding the human lipoprotein precursor protein signal peptide 14 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 lipoprotein precursor protein signal peptide 14 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 and performs endonucleation.
  • Antisense RNA and DNA and ribozymes are available Any RNA or DM synthesis technology, such as solid-phase phosphate amide chemical synthesis, has been widely used. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA.
  • This DM sequence has been integrated downstream of the RNA polymerase promoter of the vector.
  • it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human lipoprotein precursor protein signal peptide 14 can be used for the diagnosis of diseases related to human lipoprotein precursor protein signal peptide 14.
  • the polynucleotide encoding human lipoprotein precursor protein signal peptide 14 can be used to detect the expression of human lipoprotein precursor protein signal peptide 14 or the abnormal expression of human lipoprotein precursor protein signal peptide 14 in a disease state.
  • the DNA sequence encoding human lipoprotein precursor protein signal peptide 14 can be used to hybridize biopsy specimens to determine the expression status of human lipoprotein precursor protein signal peptide 14.
  • Hybridization techniques include Sou thern blotting, Nor thern blotting, and in situ hybridization.
  • Some or all of the 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 tissues.
  • Human lipoprotein precursor protein signal peptide 14 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect human lipoprotein precursor protein signal peptide 14 transcription products.
  • Human lipoprotein precursor protein signal peptide 14 gene can also be used to diagnose human lipoprotein precursor protein signal peptide 14-related diseases.
  • Human lipoprotein precursor protein signal peptide 14 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human lipoprotein precursor protein signal peptide 14 DM sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, 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.
  • an important first step is to locate these DNA sequences on a chromosome.
  • a PCR primer (preferably 15-35b P ) is prepared from the cDNA, and the sequence can be mapped on the chromosome. 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 DM 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, Mendel ian Inheritance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions. 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 diseased and oncoming 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 with 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 Figure resolution 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 lipoprotein precursor protein signal peptide 14 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human lipoprotein precursor protein signal peptide 1 4 to be 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 diagnosing physician.

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Abstract

L'invention concerne un nouveau polypeptide, une prolipoprotéine humaine 14 peptide signal, 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 prolipoprotéine humaine 14 peptide signal.
PCT/CN2000/000514 1999-11-30 2000-11-27 Nouveau polypeptide, prolipoproteine humaine 14 peptide signal, et polynucleotide codant pour ce polypeptide WO2001040299A1 (fr)

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CN 99124174 CN1297940A (zh) 1999-11-30 1999-11-30 一种新的多肽——人脂蛋白前体蛋白信号肽14和编码这种多肽的多核苷酸

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CN101619346B (zh) * 2008-07-04 2014-02-19 上海交通大学医学院附属瑞金医院 人冠心病易感基因-脂蛋白a基因拷贝数变异检测方法和试剂盒

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Title
EUR. J. BIOCHEM., vol. 129, no. 1, 1982, pages 233 - 239 *
J. BIOL. CHEM., vol. 258, no. 11, 1983, pages 7141 - 7148 *
J. BIOL. CHEM., vol. 259, no. 10, 1984, pages 6195 - 6200 *

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