WO2001094594A1 - Nouveau polypeptide, serine hydrolase humaine atp-dependante 12.2, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, serine hydrolase humaine atp-dependante 12.2, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2001094594A1
WO2001094594A1 PCT/CN2001/000724 CN0100724W WO0194594A1 WO 2001094594 A1 WO2001094594 A1 WO 2001094594A1 CN 0100724 W CN0100724 W CN 0100724W WO 0194594 A1 WO0194594 A1 WO 0194594A1
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polypeptide
polynucleotide
dependent serine
human atp
sequence
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PCT/CN2001/000724
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English (en)
Chinese (zh)
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Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc.
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Priority to AU87484/01A priority Critical patent/AU8748401A/en
Publication of WO2001094594A1 publication Critical patent/WO2001094594A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide—human ATP-dependent serine proteolytic enzyme 12.2, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide.
  • L0N1 protein In 1998, Barakat S. et al. Cloned the L0N1 protein from ma ize, which is a new member of the Lon-type proteolytic enzyme family.
  • the L0N1 protein has high similarity in protein sequence with known bacterial and human Lon proteolytic enzymes, and both have a conserved substrate-binding domain and an ATP-binding domain; and the protein and the Lon protein family
  • the other members have similar biological functions and are closely related to the respiration process of the organism in vivo, which can maintain the integrity of mitochondrial DNA, but is not a component of the cytochrome complex [Barakat S., Pearce DA. Et a l, 1998, Plant Mol Biol , 37:.
  • Lon protease family members in vivo has a broad biological functions, which can be caused by abnormal expression will result in abnormalities of mitochondrial DNA structure, And affect the function of the respiratory chain, leading to abnormal metabolism of matter and energy.
  • the N-terminus of members of the enzyme family contains a conserved ATP-binding domain that is responsible for binding to ATP in the body to hydrolyze ATP and provide the energy required for the enzyme to function; in addition, the enzyme family The members also contain the following conservative consensus sequence fragments:
  • the human ATP-dependent serine protease 12.2 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 identification in the art has been required. More human ATP-dependent serine protease 12.2 proteins involved in these processes, especially the amino acid sequence of this protein was identified. Newcomer ATP-dependent serine protease 12.2 The isolation of protein-coding genes 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 the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding for DM. Object of the invention
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a human ATP-dependent serine proteolytic enzyme 12.2.
  • Another object of the present invention is to provide a genetically engineered host cell comprising a polynucleotide encoding a human ATP-dependent serine proteolytic enzyme 12.2.
  • Another object of the present invention is to provide a method for producing human ATP-dependent serine proteolytic enzyme 12.2.
  • Another object of the present invention is to provide an antibody against the human ATP-dependent serine proteolytic enzyme 12.2 of the polypeptide of the present invention.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the human ATP-dependent serine proteolytic enzyme 12.2 of the polypeptide of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with human ATP-dependent serine proteolytic enzyme 12. 2 abnormalities. Summary of invention
  • 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 125 to 460 in SEQ ID NO: 1; and (b) a sequence having 1 to 1677 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 human ATP-dependent serine proteolytic enzyme 12.2 protein activity, which comprises utilizing a 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 ATP-dependent serine proteolytic enzyme 12.2, which includes detecting the polypeptide or a polynucleotide sequence encoding the same in a biological sample. Mutations, or the amount or biological activity of a polypeptide of the invention in a biological sample.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the preparation of a polypeptide and / or polynucleotide of the present invention for the treatment of cancer, developmental disease or immune disease or other drugs caused by abnormal expression of human ATP-dependent serine proteolytic enzyme 12. 2 use.
  • FIG. 1 is a comparison diagram of gene chip expression profiles of human ATP-dependent serine proteolytic enzyme 12.2 and human ATP-dependent serine proteolytic enzyme 48 of the present invention.
  • the upper graph is a graph of the expression profile of human ATP-dependent serine protease 12.2
  • the lower graph is the graph of the expression profile of human ATP-dependent serine protease 48.
  • 1 indicates fetal kidney
  • 2 indicates fetal large intestine
  • 3 indicates fetal small intestine
  • 4 indicates fetal muscle
  • 5 indicates fetal brain
  • 6 indicates fetal bladder
  • 7 indicates unstarved L02
  • 8 indicates L02 +, lhr, As 3+
  • 9 indicates ECV304 PMA-
  • 10 means ECV304 P A +
  • 11 means fetal liver
  • 12 means normal liver
  • 13 means thyroid
  • 14 means skin
  • 15 means fetal lung
  • 16 means lung
  • 17 means lung cancer
  • 18 means fetal spleen
  • 19 means spleen
  • 20 is the prostate
  • 21 is the fetal heart
  • 22 is the heart
  • 23 is the muscle
  • 24 is the testis
  • 25 is the fetal thymus
  • 26 is the thymus.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated human ATP-dependent serine proteolytic enzyme 12.2. 12kDa is the molecular weight of the protein. The arrow indicates the isolated protein band. Invention soluble
  • 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 a “polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural sequence related to the protein molecule. Amino acid.
  • a “variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to natural, recombinant or synthetic proteins and fragments thereof in suitable The ability to induce a specific immune response in an animal or cell and to bind to specific antibodies.
  • An "agonist” refers to a molecule that, when combined with human ATP-dependent serine proteolytic enzyme 12.2, can cause the protein to change and thereby regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to a human ATP-dependent serine proteolytic enzyme 12.2.
  • Antagonist refers to a biological activity or immunity that can block or regulate human ATP-dependent serine protease 12.2 when combined with human ATP-dependent serine protease 12.2 Chemically active molecules. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that can bind to a human ATP-dependent serine proteolytic enzyme 12.2.
  • Regular refers to a change in the function of human ATP-dependent serine protease 12.2, including an increase or decrease in protein activity, a change in binding characteristics, and any other organism of human ATP-dependent serine protease 12.2 Changes in nature, function, or immunity.
  • 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 ATP-dependent serine proteolytic enzymes using standard protein purification techniques 12.2. Substantially pure human ATP-dependent serine protease 12.2 produces a single main band on a non-reducing polyacrylamide gel. Human ATP-dependent serine proteolytic enzymes 12.2 Purity of peptides Analyze the 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 Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences based on different methods such as the Clus ter method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). 0 The Clus ter method checks all The distances arrange the groups of sequences into clusters. 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 assay may be Jotun Hein percent identity between nucleic acid sequences Clus ter or a method well known in the art (Hein J., (1990) Methods in enzymology 183: 625-645) 0
  • 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 substitution 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 HFP or a chemical modification of its nucleic acid. This chemical modification may be a substitution of a hydrogen atom with a fluorenyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? (& 1) ') 2 and?, Which specifically bind to the human ATP-dependent serine protease 12.2 epitope.
  • 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).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances in the natural state .
  • isolated human ATP-dependent serine proteolytic enzyme 12.2 refers to human ATP-dependent serine proteolytic enzyme 12.2 which is substantially free of other proteins, lipids, Sugars or other substances. Those skilled in the art can purify human ATP-dependent serine protease 12.2 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the human ATP-dependent serine proteolytic enzyme 12. 2 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human ATP-dependent serine proteolytic enzyme 12. 2, which basically consists of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptides of the present invention can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques.
  • 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 invention also includes fragments, derivatives and analogs of human ATP-dependent serine proteolytic enzyme 12.2.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the human ATP-dependent serine proteolytic enzyme 12.2 of the invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: U) a type 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 substituted
  • the amino acid may or may not be encoded by the genetic code; or ( ⁇ ) such a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or (III) such a Species, wherein the mature polypeptide is fused with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) a polypeptide sequence in which an additional amino acid sequence is fused into a mature polypeptide (such as Leader sequence or secretory sequence or the sequence or protease sequence used to purify this polypeptide).
  • such fragments, derivatives, and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 1677 bases in length and its open reading frame 125-460 encodes 111 amino acids.
  • this polypeptide has a similar expression profile to human ATP-dependent serine protease 48, and it can be deduced that the human ATP-dependent serine protease 12.2 has human ATP-dependent serine protease 48 similar features.
  • the polynucleotide of the present invention may be in the form of DM or RM.
  • DM forms include cDM, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be encoded Chain or non-coding chain.
  • 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 a denaturant during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficol l, 42 ° C, etc .; or (3) only between two sequences 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 ATP-dependent serine proteolytic enzymes 12.2.
  • 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 ATP-dependent serine proteolytic enzyme 12.2 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 genomic or cDNA libraries with probes to detect homologous polynucleosides Acid sequences, and 2) antibody screening of expression libraries to detect cloned polynucleotide fragments with common structural characteristics.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DM 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 DM sequences is often the method of choice. The more commonly used method is the separation of cDM sequences.
  • the standard method for isolating the cDM of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDM library.
  • mMA plasmid or phage cDM library.
  • kits are also commercially available (Qiagene).
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manua 1, Cold Spoon 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 can be screened from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DM-DNA or DNA-RM hybridization; ( 2 ) the presence or absence of marker gene functions; (3) determination of human ATP-dependent serine protease 12.2 transcripts (4) Detecting the protein product of gene expression by immunological techniques or measuring biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is usually a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DM probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of human ATP-dependent serine protease 12.2 gene expression can be detected using immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). .
  • a method (Saiki, et al. Science 1985; 230: 1350-1354) using PCR to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-rapid amplification of cDNA ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • Polynucleotide sequences of the gene of the present invention obtained as described above, or various DNA fragments can be used It is determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. To obtain the full-length CDM sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell genetically engineered using the vector of the present invention or directly using human ATP-dependent serine proteolytic enzyme 12.2 coding sequence, and to produce the present invention by recombinant technology Methods of the polypeptide.
  • a polynucleotide sequence encoding a human ATP-dependent serine proteolytic enzyme 12.2 may be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al.
  • any plasmid and vector can be used to construct recombinant expression vectors.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • 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 expressed by DM, 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 adenoviral enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide for selection
  • selectable marker genes to provide for selection
  • the phenotypic traits of transformed host cells such as dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E. coli.
  • GFP green fluorescent protein
  • a polynucleotide encoding a human ATP-dependent serine proteolytic enzyme 12.2 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetic engineering containing the polynucleotide or the recombinant vector.
  • Host cells refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf 9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DM sequence according to the present invention or a recombinant vector containing the DNA sequence can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryote, such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human ATP-dependent serine proteolytic enzyme 12. 2 (Scence, 1984; 224: 1431). Generally speaking, 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.
  • 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 not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic lysis, 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.
  • polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infections and immune diseases, etc. .
  • Lon-type proteolytic enzyme family catalyze the ATP-dependent degradation of mitochondrial matrix proteins.
  • the Lon protein family is closely related to the respiration process of living organisms. It can maintain the integrity of mitochondrial DNA, but it is not a component of the cytochrome complex. Abnormal expression can cause mitochondrial DM structural abnormalities, and affect the respiratory chain function, resulting in abnormal material and energy metabolism.
  • the abnormal expression of the human ATP-dependent serine proteolytic enzyme of the present invention will produce various diseases, especially mitochondrial diseases, metabolic disorders related to energy and material metabolism, and disorders of growth and development. These diseases include, but are not Limited to:
  • Organic acidemia isovaleric acidemia, propionic acidemia, methylmalonic aciduria, combined carboxylase deficiency, glutaric acid type I, etc. .
  • Amino acid metabolism defects phenylketonuria, tyrosine metabolism defects such as albinism, sulfur amino acid metabolism defects, tryptophan metabolism defects such as tryptophanemia, branch amino acid metabolism defects, glycine metabolism defects such as Glycineemia, hypersarcosineemia, proline and hydroxyproline metabolism defects, glutamate metabolism defects, urea cycle metabolism defects, histidine metabolism defects, lysine metabolism defects , And other amino acid metabolism defects.
  • Mucopolysaccharidosis and other marginal diseases Mucopolysaccharidosis ⁇ ⁇ ⁇ type, mucopolysaccharidosis marginal diseases such as rheumatoid mucopolysaccharidosis and mucolipid storage disease.
  • Purine and Pyrimidine Metabolism Defects Abnormal purine metabolism, such as Ray-niney syndrome, xanthineuria, abnormal pyrimidine metabolism, such as orotic aciduria, and adenosine deaminase deficiency.
  • Lipid metabolism abnormalities hyperlipoproteinemia, familial hyper alpha-lipoproteinemia, familial P-lipoproteinemia, familial hypo-p-lipoproteinemia, familial lecithin-cholesterol acetyltransferase Deficiency.
  • Glucose metabolism defects Congenital sugar digestion and absorption defects such as congenital lactose intolerance, hereditary fructose intolerance, monosaccharide metabolism defects such as galactosemia, fructose metabolism defects, glycogen metabolism diseases such as glycogen storage Backlog.
  • Growth and development disorders mental retardation, cerebral palsy, brain development disorders, familial cerebral nucleus dysplasia syndrome, skin, fat and muscular dysplasias such as congenital skin relaxation, premature aging, congenital horn Poor metabolism, various metabolic defects such as various amino acid metabolic defects, Syndrome, dwarfism, sexual retardation, etc.
  • Congenital malformations spina bifida, craniocerebral fissure, anencephaly deformity, cerebral bulge, foramen malforma, Down syndrome, congenital hydrocephalus, aqueduct malformation, dwarfism of cartilage hypoplasia, spinal epiphyseal dysplasia, Pseudochondral dysplasia, Langer-Giedion syndrome, funnel chest, gonad hypoplasia, congenital adrenal hyperplasia, upper urethra, cryptorchidism, short stature syndrome such as Conradi syndrome and Danbol t-Clos syndrome Syndrome, congenital glaucoma or cataract, congenital lens abnormality, congenital blepharoplasia, retinal dysplasia, congenital optic nerve atrophy, congenital sensorineural hearing loss, cracked hand and feet, teratosis, Wi ll iams synthesis Syndrome, Alagi iie Syndrome, Bezier Syndrome, etc.
  • Abnormal expression of the human ATP-dependent serine proteolytic enzyme of the present invention will also generate certain tumors, certain hereditary, hematological diseases, and immune system diseases.
  • the polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially mitochondrial disease, metabolic disorders related to energy and material metabolism, and growth and development disorders. Diseases, congenital malformations, certain tumors, certain hereditary, hematological and immune system diseases, etc.
  • the invention also provides methods of screening compounds to identify agents that increase (agonist) or suppress (antagonist) human ATP-dependent serine proteolytic enzymes 12.2.
  • Agonists enhance human ATP-dependent serine proteolytic enzymes.
  • 12.2 Stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • membrane preparations of mammalian cells or expressing human ATP-dependent serine proteolytic enzyme 12.2 with labeled human ATP-dependent serine proteolytic enzyme 12.2 can be cultured in the presence of drugs. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human ATP-dependent serine proteolytic enzymes 12.2 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonist of human ATP-dependent serine protease 12.2 can bind to human ATP-dependent serine protease 12.2 and eliminate its function, or inhibit the production of the polypeptide, or with the active site of the polypeptide Binding prevents the polypeptide from functioning biologically.
  • human ATP-dependent serine proteolytic enzyme 12.2 When screening compounds as antagonists, human ATP-dependent serine proteolytic enzyme 12.2 can be added to the bioanalytical assay, and by measuring the compounds against human ATP-dependent serine proteolytic enzyme 12.2 and their receptors, Effect to determine whether a 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 ATP-dependent serine protease 12.2 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, human ATP-dependent serine protease 12.2 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 against human ATP-dependent serine proteolytic enzyme 12.2 epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced using human ATP-dependent serine proteolytic enzyme 12.2 by direct injection of 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 Freund's adjuvant and the like.
  • Techniques for preparing monoclonal antibodies to human ATP-dependent serine proteolytic enzyme 12. 2 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human B -Cell hybridoma technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions to non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851).
  • the existing technology for producing single chain antibodies U.S. Pat No. 4946778, can also be used to produce single chain antibodies against human ATP-dependent serine proteolytic enzymes 12.2.
  • Antibodies against human ATP-dependent serine protease 12.2 can be used in immunohistochemical techniques to detect human ATP-dependent serine protease 12.2 in biopsy specimens.
  • Monoclonal antibodies that bind to human ATP-dependent serine protease 12.2 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 against a specific bead site in 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 human ATP-dependent serine protease 12 . 2 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human ATP-dependent serine proteolytic enzymes 12.2. Administration of appropriate doses of antibodies can stimulate or block the production or activity of human ATP-dependent serine protein hydrolase 12.2.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human ATP-dependent serine proteolytic enzyme 12.2 levels.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human ATP-dependent serine protease 12.2 detected in the test can be used to explain the importance of human ATP-dependent serine protease 12.2 in various diseases and to diagnose human ATP-dependent serine Diseases where proteolytic enzymes 12.2 work.
  • polypeptides of the present invention can also be used for peptide mapping, for example, the polypeptides can be physically, chemically or enzymatically Specific cleavage and one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, preferably mass spectrometry.
  • Polynucleotides encoding human ATP-dependent serine proteolytic enzyme 12.2 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 ATP-dependent serine proteolytic enzyme 12.2. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human ATP-dependent serine protease 12.2 to inhibit endogenous human ATP-dependent serine protease 12.2 activity.
  • a variant human ATP-dependent serine protease 12.2 may be a shortened human ATP-dependent serine protease 12.2 that lacks a signaling domain, although it can bind to downstream substrates, However, it lacks signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of human ATP-dependent serine proteolytic enzyme 12.2.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding human ATP-dependent serine protease 12.2 into a cell.
  • recombinant viral vectors carrying a polynucleotide encoding a human ATP-dependent serine proteolytic enzyme 12.2 can be found in the existing literature (Sambrook, et al.). Alternatively, a recombinant polynucleotide encoding human ATP-dependent serine proteolytic enzyme 12.2 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 RM and DNA
  • ribozymes that inhibit human ATP-dependent serine protease 12.2 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RM molecule that can specifically decompose a specific RM. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA and performs endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or MA synthesis techniques, such as solid-phase phosphate amide chemical synthesis to synthesize oligonucleotides.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RM. This DM sequence has been integrated downstream of the RM polymerase promoter of the vector. 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 phosphorothioate or peptide bond instead of the phosphodiester bond is used for the ribonucleoside linkage.
  • the polynucleotide encoding human ATP-dependent serine protease 12.2 can be used for the diagnosis of diseases related to human ATP-dependent serine protease 12.2.
  • the polynucleotide encoding human ATP-dependent serine protease 12.2 can be used to detect the expression of human ATP-dependent serine protease 12.2 or human ATP-dependent serine protease 12.2 Abnormal expression.
  • a DNA sequence encoding human ATP-dependent serine protease 12.2 can be used to hybridize biopsy specimens to determine the expression of human ATP-dependent serine protease 12.2.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization.
  • 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.
  • a microarray or a DNA chip also referred to as a "gene chip”
  • Human ATP-dependent serine protease 12.2 specific primers for MA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect human ATP-dependent serine protease 12.2 transcripts.
  • Human ATP-dependent serine protease 12.2 mutations can also be used to diagnose human ATP-dependent serine protease 12.2 related diseases.
  • Human ATP-dependent serine protease 12.2 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type human ATP-dependent serine protease 12.2 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. Therefore, Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.
  • a PCR primer (preferably 15-35bp) is prepared from the cDNA, and the sequence can be located on the chromosome. 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 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 hybrid pre-selection to construct chromosome-specific cDM libraries.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is 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 chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the 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 reminders authorize them to be administered to the body by government agencies that manufacture, use, or sell them.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human ATP-dependent serine protease 12.2 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dose range of human ATP-dependent serine proteolytic enzyme 12.2 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician. Examples
  • Total RM of human fetal brain was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) m 2ug poly (A) mRNA was isolated from total IWA using Quik mRNA Isolat ion Kit (product of Qiegene) to form cDNA by reverse transcription.
  • the Smart cDNA cloning kit purchased from Clontech was used to insert the cDNA fragments 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
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0385e08 was a new D.
  • the inserted cDNA fragments contained in this clone were determined in both directions by synthesizing a series of primers.
  • CDNA was synthesized using fetal brain total RNA as a template and ol igo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, the following primers were used for PCR amplification:
  • Primer2 5'- ATGTGGAGTGGGAAATCAGGGGAC-3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp of the 5th end of SEQ ID NO: 1;
  • Primer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.
  • a reaction volume of 50 ⁇ 1 contains 50 mmol / L KCl, 10 mmol / L Tris-HCl pH 8. 5, 1. 5 mmol / L MgCl 2 , 20 ( ⁇ mol / L dNTP, 1 Opmol primer, . 1U Taq DNA polymerase (Clontech Co.) on the PE9600 DNA thermal cycler (Perkin-Elmer Co.) by the reaction of 25 cycles of the following conditions: 94 ° C 30sec; 55.C 30sec ; 72 ° C 2min 0 in During RT-PCR, ⁇ -act in was used as a positive control and template blank was used as a negative control.
  • the amplified product was purified using a QIAGEN kit and ligated to a pCR vector using a TA cloning kit (Irivi trogen).
  • MA sequence The analysis results showed that the DNA sequence of the PCR product was exactly the same as l-1677bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human ATP-dependent serine protease 12.2 gene expression
  • RNA extraction in one step [Anal. Biochera 1987, 162, 156-159] 0
  • This method includes acid sulfur Guanidinium cyanate phenol-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1), 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.
  • Electrophoresis was performed on a 1.2 ⁇ g agarose gel containing 2 ⁇ g RA, containing 20 raM 3- (N-morphino) propanesulfonic acid (PH7.0) -5 mM sodium acetate-ImM EDTA-2. 2M formaldehyde. then transferred to nitrocellulose by a -. 32 P dATP labeled probe 32 P- DM prepared by the random primer SYSTEM DNA probes used for PCR amplification shown in FIG human ATP-dependent serine protease. Hydrolase I 2 ⁇ 2 coding region sequence (125bp to 460bp).
  • a 32P-labeled probe (approximately 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred in a solution at 42 ° C. Overnight, the solution contained 50 ° C. Formamide-25 mM KH 2 PO 4 (pH 7.4)-5 x SSC-5 x Denhardt, s solution and 200 g / ml salmon sperm DNA. After hybridization, the filter was set at 1 x SSC-0. 1% SDS was washed at 55 ° C for 30 min. Then, analysis and quantification were performed using Phosphor Imager.
  • Example 4 In vitro expression, isolation and purification of recombinant human ATP-dependent serine protease 12.2 according to SEQ ID NO: 1 and the coding region sequence shown in Figure 1, a pair of specific amplification primers were designed, the sequence is as follows:
  • Pr imer 3 5'-CCCCATATGATGAGCCAGATGCTGTGGAAGAGA-3 '(Seq ID No: 5)
  • Pr imer4 5'-CATGGATCCTTACATCCAGGCATTATTGCCAGC-3' (Seq ID No: 6)
  • the two ends of these two primers contain Ndel and BamHI digestion respectively Site, followed by the coding sequence of the 5 ,, and 3 'ends of the gene of interest, respectively.
  • the Ndel and BamHI restriction sites correspond to the expression vector plasmid pET- 28 b (+) (Novagen, Cat. No. 69865. 3 Selective endonuclease sites on).
  • the PCR reaction was performed using pBS-0385e08 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were: 'The total volume of 50 ⁇ 1 contains 10 pg of pBS-0385eO8 plasmid, the primers Pr imer-3 and Pr imer-4 are l Opmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1, respectively.
  • Cycle parameters 94 ° C 20s, 60 ° C 30s, 68 ° C 2 rain, 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 ligated product was transformed with colibacillus DH5 0C by the calcium chloride method. After being cultured overnight in LB plates containing kanamycin (final concentration 30 ⁇ ⁇ / ⁇ 1), positive colonies were screened by colony PCR method and sequenced. A positive clone (pET-0385e08) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) P lySs (product of Novagen) using the calcium chloride method.
  • a peptide synthesizer (product of PE company) was used to synthesize the following human ATP-dependent serine protease 12.2 specific peptides:
  • the polypeptide is coupled with hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin For the method, see: Avraraeas, et al. Immunochemi s try, 1969; 6: 43. Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex and complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex and incomplete Freund's adjuvant were used to boost the immunity once.
  • a titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum.
  • Protein A-Sepharose was used to isolate total IgG from antibody-positive rabbit serum.
  • the peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography.
  • the immunoprecipitation method demonstrated that the purified antibody specifically binds to human ATP-dependent serine proteolytic enzyme 12.2.
  • Example 6 Application of the polynucleotide fragment of the present invention as a hybridization probe
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by using a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods. They all use the same steps of hybridization after fixing the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer, so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthetic polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes, and Incubation hybridizes the probe to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment utilizes 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 from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • the GC content is 30% -70%, and the non-specific hybridization increases when it exceeds;
  • 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 For homology comparison of the regions, 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 generally;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (bond):
  • Probe 2 which belongs to the second type of probe, is equivalent to the replacement mutant sequence of the gene fragment of SEQ ID NO: 1 or its complementary fragment (41Nt):
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • Two NC membranes are required for each probe, so that they can be used in the following experimental steps.
  • the film was washed with high-strength conditions and strength conditions, respectively. .
  • the sample membrane was placed in a plastic bag, and 3-1 Omg pre-hybridization solution (lOxDenhardt-s; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA)) was added. After closing the bag, 68. C water bath for 2 hours.
  • 3-1 Omg pre-hybridization solution lOxDenhardt-s; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DNA)
  • High-intensity washing film 1) Take out the hybridized sample membrane.
  • Gene chip or DNA microarray is a new technology that many national laboratories and large pharmaceutical companies are currently developing and developing. 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 fast, 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 DeRis i, JL, Lyer, V. & Brown, PO (1997) Science 278, 680-686. And Hel le, RA, Schema, M. Chai, A., Shalom, D., (1997) PNAS 94: 2150-2155.
  • a total of 4,000 polynucleotide sequences of various full-length cDs are used as target DMs, including the polynucleotides of the present invention. Amplify them separately by PCR, and adjust the concentration of the amplified products to At about 500ng / ul, a Cartesian 7500 spotter (purchased from Cartesian Company, USA) was used to point on the sloped glass medium, and the distance between the points was 280 ⁇ m. The spotted slides were hydrated and dried, cross-linked in a UV cross-linker, and dried after elution to fix the DNA on the glass slide to prepare a chip. The specific method steps have been reported in the literature. The sample post-processing steps in this embodiment are:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and the mRNA was purified with Oligotex mRNA Midi Ki t (purchased from QiaGen).
  • Cy3dliTP (5-Amino-propargyl-2'-deoxyuridine 5'-tr iphate coupled to Cy3 f luorescent dye, purchased from Araersham Phamacia Biotech) was used to label the mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP (5-Amino-propargyl-2 '-deoxyuridine 5'-triphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech, labeled the body's specific tissue (or stimulated cell line) mRNA, and purified the probe to prepare a probe.
  • Cy5dUTP 5-Amino-propargyl-2 '-deoxyuridine 5'-triphate coupled to Cy5 fluorescent dye
  • the probes from the above two tissues and the chip were respectively hybridized in a UniHyb TM Hybridizat ion Solut ion (purchased from TeleChem) hybridization solution for 16 hours, and washed with a washing solution (lx SSC, 0.2% SDS) at room temperature. Scanning was performed with a ScanArray 3000 scanner (purchased from General Scanning, USA), and the scanned images were analyzed by Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, L02 cell line stimulated by arsenic for 1 hour, L02 cell line stimulated by arsenic for 6 hours prostate, heart, lung Cancer, fetal bladder, fetal small intestine, fetal large intestine, fetal thymus, fetal muscle, fetal liver, fetal kidney, fetal spleen, fetal brain, fetal lung, and fetal heart.

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Abstract

L'invention concerne un nouveau polypeptide, une sérine hydrolase humaine ATP-dépendante 12.2, et un polynucléotide codant 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 la sérine hydrolase humaine ATP-dépendante 12.2.
PCT/CN2001/000724 2000-05-09 2001-05-08 Nouveau polypeptide, serine hydrolase humaine atp-dependante 12.2, et polynucleotide codant ce polypeptide WO2001094594A1 (fr)

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CN 00115605 CN1322832A (zh) 2000-05-09 2000-05-09 一种新的多肽——人atp依赖的丝氨酸蛋白水解酶12.2和编码这种多肽的多核苷酸
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0887414A2 (fr) * 1997-06-09 1998-12-30 Smithkline Beecham Plc Sérine protéases humaines HGBAB90
WO1999040183A1 (fr) * 1998-02-06 1999-08-12 Human Genome Sciences, Inc. Serine-protease humaine et polypeptides serpin

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0887414A2 (fr) * 1997-06-09 1998-12-30 Smithkline Beecham Plc Sérine protéases humaines HGBAB90
WO1999040183A1 (fr) * 1998-02-06 1999-08-12 Human Genome Sciences, Inc. Serine-protease humaine et polypeptides serpin

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