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WO2001049861A1 - Nouveau polypeptide, spase humaine i 13, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, spase humaine i 13, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001049861A1
WO2001049861A1 PCT/CN2000/000648 CN0000648W WO0149861A1 WO 2001049861 A1 WO2001049861 A1 WO 2001049861A1 CN 0000648 W CN0000648 W CN 0000648W WO 0149861 A1 WO0149861 A1 WO 0149861A1
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Prior art keywords
polypeptide
spase
human
polynucleotide
enzyme
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PCT/CN2000/000648
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Fudan University
Shanghai Bio Door Gene Technology Ltd.
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Priority to AU21422/01A priority Critical patent/AU2142201A/en
Publication of WO2001049861A1 publication Critical patent/WO2001049861A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention belongs to the field of biotechnology, and specifically, the present invention describes a new polypeptide- ⁇ SPase I enzyme 13, and a polynucleotide sequence encoding the polypeptide.
  • the invention also relates to methods and applications for preparing such polynucleotides and polypeptides.
  • SPases Signal peptidases
  • Lep lead peptidases
  • E. coli guide peptidase [Lep (EC)], Typhoid Typhimurium [Lep (ST)], mitochondrial inner membrane protease I, II (IMP1 and IMP2) of Saccharomyces cerevisiae belong to Spases I.
  • Signal peptidases that remove signal peptides in eukaryotic cells exist as oligomers, contain at least five subunits, and are localized on the endoplasmic reticulum membrane.
  • the two subunits of the mammalian signal peptidase complex (SPC), SPC18 and SPC21, and the SEC11 subunit of yeast have similar structures to the SPases I enzymes of prokaryotes.
  • All signal peptidase data shows that it is a membrane bulk protein.
  • Lep EC
  • the gene map of the catalytic region shows that the protein contains 5 recognizable domains, which are: 2 transmembrane regions ( HI, H2), a weaker hydrophobic region (H3) and two polar regions (PI, P2), and both the amino and C-termini face the periplasm.
  • the HI-P1 region is mostly unrelated to signal peptidase activity, and the replacement of H2 with unrelated hydrophobic amino acids has little effect on the activity, proving that its active site is located in the periplasmic domain.
  • SPases are highly specific for substrates. Three domains in the signal peptide are conserved: the amino-terminal domain (n-region), consisting of 1-5 amino acids, at least one positively charged amino acid; the central hydrophobic region (h-region), consisting of 7- 15 amino acids; more polar C-region (c-region), consisting of 3-7 amino acids, and is the site of action of SPase.
  • n-region amino-terminal domain
  • h-region central hydrophobic region
  • c-region more polar C-region
  • the smallest substrate that can be excised by Lep (EC) is ALA KI, but the more efficient substrate is FSASALAIKI containing a hydrophobic region, where i represents the cleavage site.
  • the surfactant Triton X-100 can accelerate the catalytic activity of lead peptidase, and it has also been found that phospholipids can stimulate the activity of lead peptidase. It is possible that phospholipids may also play an important role in the catalytic mechanism.
  • Lep has an amino acid sequence of 93 ° / with P. fluorescens and subtilase, respectively. , 50% and 31% homology, mitochondrial Impl and Subtilase has 28% homology, and bacterial signal peptidases have 20 with SEC11, SPC18 and SPC21 subunits of eukaryotic cells. -30% homology.
  • Serase SP90 is the only Ser that is conserved and related to activity. However, compared with other Ser protease families, it does not have His as a proton donor and acceptor for activation.
  • the signal peptidase complex of eukaryotic cells is much more complicated than the guide peptidase in bacteria, but it is closest to the Ser-type peptidase. Sequence alignment studies have shown that the signal peptidase catalytic activity of eukaryotic cells depends on the Ser / His dyad, and not on Ser / Lys like SPase I.
  • the human SPase I enzyme 13 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 SPase I enzyme 13 proteins involved in these processes. In particular, the amino acid sequence of this protein is identified. Isolation of the new SPase I enzyme 13 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 the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding for DM.
  • 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 SPase I enzyme 13.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human SPase I enzyme 13.
  • Another object of the present invention is to provide a method for producing human SPase I enzyme 13.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention, ⁇ SPase I enzyme 13.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors of human SPase I enzyme 13 against the polypeptide of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of human SPase I enzyme 13.
  • 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 297-653 in SEQ ID NO: 1; and (b) a sequence having 1-752 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 SPase I enzyme 13 protein, which comprises utilizing the polypeptide of the invention.
  • the present invention also relates to a compound obtained by the 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 SPase I enzyme 13 protein, which comprises detecting the polypeptide or a polynucleoside encoded therein in a biological sample. Mutations in the acid sequence, 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 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 SPase I enzyme 13.
  • FIG. 1 is a comparison diagram of the amino acid sequence homology of the SPase I enzyme 13 of 54 amino acids and the domain SPase I enzyme at 56-110 of the present inventor.
  • the upper sequence is human SPase I enzyme 13 and the lower sequence is SPase I enzyme Domain.
  • ⁇ "and”: “and”. “Indicate that the probability of the same amino acid appearing between two sequences decreases in sequence.
  • FIG. 2 is a polyacrylamide gel electrophoresis image (SDS-PAGE) of the isolated human SPase 1 enzyme 13.
  • FIG. 13kDa is the molecular weight of the protein. The arrow indicates the isolated protein band.
  • Nucleic acid sequence refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and may also refer to the genome or synthetic DNA or RM, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a 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 means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that, when combined with human SPase I enzyme 13, causes a change in the protein to regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that binds human SPase I enzyme 13.
  • Antagonist refers to a molecule that, when combined with human SPase I enzyme 13, can block or regulate the biological or immunological activity of human SPase I enzyme 13.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that binds human SPase I enzyme 13.
  • “Regulation” refers to a change in the function of human SPase I enzyme 13, including an increase or decrease in protein activity, Changes in binding properties and any other biological, functional or immune properties of human SPase I enzyme 13.
  • “Substantially pure” means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify human SPase I enzyme 13 using standard protein purification techniques. The substantially pure human SPase I enzyme 13 produces a single main band on a non-reducing polyacrylamide gel. The purity of human SPase I enzyme 13 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of multiple nucleotides through 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 can 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 or Northern 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 as a specific or selective interaction.
  • 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 by the MEGALIGN program (Lasergene software package, DNASTAR, Inc., Madison Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Clus ter method (Higgins, D. G. and P. M. Sharp (1988) Gene 73: 237-244). The Clus ter method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by:
  • the percent identity between nucleic acid sequences can also be determined by the Clus ter method or by methods known in the art such as Jotun Hein (Hein J., (1990) Methods in enzymology 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 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 RM sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to the "sense strand”.
  • Derivative refers to HFP or a chemical modification of its nucleic acid. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody means a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and?, Which specifically bind to the epitope of human SPase I enzyme 13.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of matter from its original environment (for example, its natural environment if it is naturally occurring).
  • a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist 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 SPase I enzyme 13 means that human SPase I enzyme 13 is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify human SPase I enzyme 13 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 SPase I enzyme 13 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human SPase I enzyme 13, which consists essentially 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. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.
  • the invention also includes fragments, derivatives and analogs of human SPase I enzyme 13.
  • fragments, derivatives and analogs of human SPase I enzyme 13 As used in the present invention, the terms “fragment”, “derivative” and “analog” refer to substantially maintaining the human SPase I enzyme 13 phase of the present invention 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 a genetic codon; or ( ⁇ ) a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or ( ⁇ ⁇ )
  • Such a type in which the mature polypeptide is fused with another compound such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol
  • (IV) a type in which the additional amino acid sequence is fused into the mature polypeptide and the polypeptide sequence is formed (Such as the leader or secretory sequence or the sequence used to purify this polypeptide or protein sequence).
  • 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 a 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 752 bases, and its open reading frame (297-653) encodes 118 amino acids. This peptide has the characteristic sequence of the SPase I enzyme, and it can be deduced that the human SPase I enzyme 13 has the structure and function represented by the SPase I enzyme.
  • the polynucleotide of the present invention may be in the DM form or the RM form.
  • DM forms include cDNA, genomic DNA, or synthetic DM.
  • DNA can be single-stranded or double-stranded.
  • the DM can be a coding chain or a 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 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.
  • Variants of this polynucleotide may 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 (with at least two sequences between
  • 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, 60 ° C; or (2) Add denaturants 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 SPase I enzyme 13.
  • 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 SPase I enzyme 13 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 CDM libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DM sequence from the DM of the genome; 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 isolation of cDNA 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 cDNA library.
  • Various methods have been developed for raRNA extraction, and kits are also commercially available (Qi agene). And the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spiring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (1) DM_DM or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) measuring the level of human SPase I enzyme 13 transcripts; (4) by immunization Technology or measurement of biological activity to detect gene-expressed protein products. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 Nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is 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).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect protein products expressed by the human SPase I enzyme 13 gene.
  • ELISA enzyme-linked immunosorbent assay
  • a method (Saiki, et al. Science 1985; 230: 1350-1354) using PCR technology to amplify DNA / MA 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 / RM 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 DM fragments and the like obtained as described above can be measured by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising a polynucleotide of the present invention, and a host cell genetically engineered using the vector of the present invention or directly using a human SPase I enzyme 13 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology.
  • a polynucleotide sequence encoding human SPase I enzyme 13 can be inserted into a vector to form a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, bacteriophages, 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 a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain origins of replication, promoters, marker genes, and translational regulatory elements.
  • Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human SPase I enzyme 13 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manual, Colling Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRM synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors 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 phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human SPase I enzyme 1 3 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • 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.
  • Escherichia coli, Streptomyces bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells insect cells
  • fly S2 or Sf 9 animal cells
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the (3 ⁇ 4 (12 ) method. The steps used are well known in the art. Alternatively, MgCl 2 can be used. If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DM transfection methods can be selected: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and lipid Body packaging, etc.
  • polynucleotide sequence of the present invention can be used to express or produce recombinant human SPase I enzyme 13 (Science, 1984; 224: 1431). Generally there are the following steps:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid
  • polypeptides of the present invention can be directly used in the treatment of diseases, for example, they can be used to treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immunological diseases.
  • SPases Signal peptidases
  • Lep lead peptidases
  • Signal peptidases that remove signal peptides in eukaryotic cells exist as oligomers, contain at least five subunits, and are localized on the endoplasmic reticulum membrane.
  • the two subunits of the mammalian signal peptidase complex (SPC), SPC18 and .SPC21, and the SEC11 subunit of yeast have similar structures to the SPases I enzymes of prokaryotic cells, and they are membrane bulk proteins.
  • the surfactant Triton X-100 can accelerate the catalytic activity of lead peptidase, and also found that phospholipids can stimulate the activity of lead peptidase. It is possible that phospholipids may also play an important role in the catalytic mechanism.
  • the polypeptide of the present invention contains a specific conserved sequence necessary for SPase I activity mot if. It can be seen that the abnormal expression of the specific signal peptidase I mot if will cause the function of the polypeptide containing the mot if of the present invention to be abnormal, resulting in the abnormal digestion of the signal peptide, and further, the signal transmission of the cell will be wrong, and May cause related diseases such as tumors, embryonic developmental disorders, growth and development disorders, inflammation, etc.
  • the abnormal expression of the human SPase I enzyme 13 of the present invention will produce various diseases, especially various tumors, embryonic developmental disorders, growth disorders, and inflammation.
  • diseases include, but are not limited to, embryonic developmental disorders : Congenital abortion, cleft palate, limb atrophy, limb differentiation disorder, hyaline membrane disease, atelectasis, polycystic kidney, double ureter, cryptorchidism, congenital inguinal hernia, double uterus, vaginal atresia, hypospadias, amphoteric deformity , Atrial septal defect, Ventricular septal defect, Pulmonary stenosis, Arterial duct occlusion, Neural tube defect, Congenital hydrocephalus, Iris defect, Congenital cataract, Congenital glaucoma or cataract, Congenital deafness Growth and development disorders: mental retardation, cerebral palsy, mental retardation, mental retardation, familial cerebellar dysp
  • Tumors of various tissues gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioblastoma , Colon cancer, melanoma, adrenal cancer, bladder cancer, bone cancer, osteosarcoma, myeloma, bone marrow cancer, brain cancer, uterine cancer, endometrial cancer, gallbladder cancer, colon cancer, thymic tumor, nasal cavity and sinus tumor, Nasopharyngeal carcinoma, laryngeal carcinoma, tracheal tumors, fibromas, fibrosarcomas, lipomas, liposarcomas, leiomyomas
  • Inflammation allergic reaction, bronchial asthma, allergic pneumonia, adult respiratory distress syndrome, sarcoidosis, rheumatoid arthritis, rheumatoid arthritis, osteoarthritis, cholecystitis, glomerulonephritis, immunity Complex glomerulonephritis, acute anterior uveitis, dermatomyositis, urticaria, atopic dermatitis, hemochromatosis, polymyositis, Addison's disease, chronic active hepatitis, intestinal emergency syndrome, Atrophic gastritis, systemic lupus erythematosus, myasthenia gravis, cerebrospinal multiple sclerosis, Guillain-Barre syndrome, intracranial granulolysis, pancreatitis, myocarditis, and infectious inflammation
  • Abnormal expression of the human SPase I enzyme 13 of the present invention will also produce certain hereditary, hematological and immune system diseases.
  • polypeptides of the present invention can be directly used in the treatment of diseases.
  • they can treat various diseases, especially various tumors, embryonic development disorders, growth disorders, inflammation, Some hereditary, hematological and immune system diseases.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human SPase I enzyme 13.
  • Agonists enhance human SPase I enzyme 13 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 SPase I enzyme 13 can be cultured together with labeled human SPase I enzyme 13 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human SPase I enzyme 13 include antibodies, compounds, receptor deletions, and analogs that have been screened. Antagonists of human SPase I enzyme 13 can bind to human SPase I enzyme 13 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions.
  • human SPase I enzyme 13 When screening compounds as antagonists, human SPase I enzyme 13 can be added to a bioanalytical assay to determine whether a compound is an antagonist by measuring the effect of the compound on the interaction between human SPase I enzyme 13 and its receptor. Antagonists can be screened in the same way as above for screening compounds Acting receptor deletions and analogs. Polypeptide molecules capable of binding to human SPase I enzyme 13 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 SPase I enzyme 13 molecule should generally be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against human SPase I enzyme 13 epitopes. These antibodies include (but are not limited to): Doklon antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, and fragments from Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting human SPase I enzyme 13 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant.
  • Techniques for preparing monoclonal antibodies to human SPase I enzyme 13 include, but are not limited to, hybridoma technology (Kohler and Mistein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-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). 0
  • Existing techniques for producing single-chain antibodies can also be used to produce single chain antibodies against human SPase I enzyme 13.
  • Anti-human SPase I enzyme 13 antibodies can be used in immunohistochemical techniques to detect human SPase I enzyme 13 in biopsy specimens.
  • Monoclonal antibodies that bind to human SPase I enzyme 13 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • human SPase I enzyme 13 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 SPase I enzyme 13 positive cells.
  • the antibodies in the present invention can be used to treat or prevent diseases related to human SPase I enzyme 13.
  • Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human SPase I enzyme 13.
  • the present invention also relates to a diagnostic test method for quantitative and localized detection of human SPase I enzyme 13 levels. These tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human SPase I enzyme 13 detected in the test can be used to explain the importance of human SPase I enzyme 13 in various diseases and to diagnose diseases in which human SPase I enzyme 13 functions.
  • the polypeptide of the present invention can also be used for peptide mapping analysis. For example, the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • Polynucleotides encoding human SPase I enzyme 13 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 SPase I enzyme 13.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant human SPase I enzyme 13 to inhibit endogenous human SPase I enzyme 13 activity.
  • a mutant human SPase I enzyme 13 may be a shortened human SPase I enzyme 13 lacking a signaling domain, and although it can bind to downstream substrates, it lacks signaling activity.
  • the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human SPase I enzyme 13.
  • Virus-derived expression vectors such as retroviruses, adenoviruses, adenovirus-associated viruses, herpes simplex virus, and parvoviruses can be used to transfer polynucleotides encoding human SPase I enzyme 13 into cells.
  • Methods for constructing recombinant viral vectors carrying a polynucleotide encoding human SPase I enzyme 13 can be found in the existing literature (Sambrook, et al.).
  • a recombinant polynucleotide encoding human SPase I enzyme 13 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 SPase I enzyme 13 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like MA molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RM to perform endonucleation.
  • Antisense RNA, DM, and ribozymes can be obtained using any existing RNA or DM 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 RNA.
  • This DNA sequence has been integrated downstream of the RNA polymerase promoter of the vector.
  • it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphorothioate or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human SPase I enzyme 13 can be used for the diagnosis of diseases related to human SPase I enzyme 13.
  • the polynucleotide encoding human SPase I enzyme 13 can be used to detect the expression of human SPase I enzyme 13 or the abnormal expression of human SPase I enzyme 13 in a disease state.
  • the DNA sequence encoding human SPase I enzyme 13 can be used to hybridize biopsy specimens to determine the expression of human SPase I enzyme 13.
  • Hybridization techniques include Southern blotting, Northern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • One of the polynucleotides of the invention Part or all can be used as probes to be fixed on a microarray or a DNA chip (also known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Human SPase I enzyme 13 specific primers can also be used to detect the transcription products of human SPase I enzyme 13 by performing RM-polymerase chain reaction (RT-PCR) in vitro amplification.
  • Detection of mutations in the human SPase I enzyme 13 gene can also be used to diagnose human SPase I enzyme 13-related diseases.
  • Human SPase I enzyme 13 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human SPase I enzyme 13 DNA 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, so Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DM sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the CDM or genomic sequence differences 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 individual, The mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable using cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human SPase I enzyme 13 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human SPase I enzyme 13 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.
  • Total RM of human fetal brain was extracted by one step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using Quik mRNA Isolat ion Kit (product of Qiegene). 2 ug poly (A) mRNA is formed into cDNA by reverse transcription. A Smart cDNA cloning kit (purchased from Clontech
  • Dye terminate cycle react ion sequencing Kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequences were compared with the public DNA sequence database (Genebank) When compared, one of the clones was found The 1176H09 cDNA sequence is new DNA.
  • a series of primers were synthesized to perform bidirectional determination of the inserted CDM fragments contained in this clone.
  • the sequence of the human SPase I enzyme 13 of the present invention and the protein sequence encoded by the protein were analyzed using the prof i le scan program (Basic local al ignment search tool) in GCG [Al tschul, SF et al. J. Mol. Biol. 1990; 215: 403-10], performing domain analysis in databases such as Prote.
  • the human SPase I enzyme 13 of the present invention is homologous to the domain SPase I enzyme at 56-110, and the homology result is shown in Fig. 1.
  • the homology rate is 0.34, the score is 18. 48, and the threshold is 17.56.
  • Example 3 Cloning of a gene encoding human SPase I enzyme 13 by ri-PCR
  • CDNA was synthesized using fetal brain cell 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:
  • Primerl 5 '— CGAGACAGAGTCTCACTCTGTTG—3' (SEQ ID NO: 3)
  • Primer2 5'- ATGAGGATAATGATACAGTTACC -3 '(SEQ ID NO: 4)
  • Pr imerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification conditions 50 ⁇ l / L KC 1, 10 mraol / L Tris-HCl, pH 8.5, 1.5 mmol / L MgCl 2 , 20 ( ⁇ mol / L dNTP) in a reaction volume of 50 ⁇ 1 , lOpmol primer, 1U Taq DNA polymerase (product of Clontech). 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. Set ⁇ -act in as a positive control and template blank as a negative control at the same time during RT-PCR.
  • the amplification products were purified using a QIAGEN kit, and linked to a pCR vector using a TA cloning kit (Invitrogen product).
  • the DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as the 1-752bp shown in SEQ ID NO: 1.
  • Example 4 Northern blot analysis of human SPase I enzyme 13 gene expression
  • RNA was used for electrophoresis on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (PH7. 0)-5 mM sodium acetate-linM EDTA-2.2 M formaldehyde . It was then transferred to a nitrocellulose membrane.
  • a 32 P dATP was used to prepare 32 P-labeled DM probes by random primer method.
  • the DM probe used was the PCR amplified human SPase I enzyme 13 coding region sequence (297bp to 653bp) shown in FIG.
  • the 32P- labeled probes (about 2 x l0 6 cpm / ml) and transferred to nitrocellulose membrane 42 in the RM is a solution.
  • the solution contains 50% formamide-25mM KH 2 PO 4 (pH 7.4) -5 x SSC-5 x Denhardt's solution and 20 ( ⁇ g / ml salmon sperm DNA.
  • the filter was placed in 1 ⁇ SSC-0. 1% SDS was washed at 55 ° C for 30 minutes. Then, Phosphor Imager was used for analysis and quantification.
  • Example 5 In vitro expression, isolation and purification of recombinant human SPase I enzyme 13
  • Pr imer 3 5'- CCCCATATGATGTCAGGTAGCAGGATTTGTGGAG -3 '(Seq ID No: 5)
  • Pr iraer4 5'- CATGGATCCTCAGCAAGTCAAGAGATTGAGACC -3' (Seq ID No: 6)
  • the restriction sites for Mel and BamHI correspond to the expression vector plasmid pET- 2 8b (+) (Novagen, Cat. No. 69865. 3 Selective endonuclease sites on).
  • PCR reaction was performed using pBS-1176H09 plasmid containing the full-length target gene as a template.
  • PCR reaction conditions were: 1 in a total volume of 50 ⁇ plasmid pBS- 1176H09 containing 10pg, Primer-3 and Primer Primer- 4 are l Opmol, Advantage polymerase Mix (Clontech Products) 1 ⁇ 1.
  • Cycle parameters 94. C 20s, 60. C 30s, 68. C 2 min, a total of 25 cycles.
  • Ndel and 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 E.
  • the following peptides specific for human SPase I enzyme 13 were synthesized using a peptide synthesizer (product of PE company): NH 2 -Met-Ser-Gly-Ser-Arg-I le-Cys-Gly-Ala-Asn-Gln-Leu- Lys-Gln-Gln-COOH (SEQ ID NO: 7).
  • the polypeptide is coupled with hemocyanin and bovine serum albumin to form a complex, respectively.
  • 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 the labeled probe and incubated to hybridize 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. Needle-to-sample hybridization has the strongest specificity and is 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
  • 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 generally 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 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
  • the sample membrane was placed in a plastic bag, and 3-10 mg of prehybridization solution (lOxDenhardt's; 6xSSC, 0.1 mg / ral CT DM (calf thymus DNA)) was added. After closing the bag, 68. C water bath for 2 hours.
  • prehybridization solution lOxDenhardt's; 6xSSC, 0.1 mg / ral CT DM (calf thymus DNA)

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Abstract

L'invention concerne un nouveau polypeptide, une SPase humaine I 13, 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 SPase humaine I 13.
PCT/CN2000/000648 1999-12-29 2000-12-25 Nouveau polypeptide, spase humaine i 13, et polynucleotide codant pour ce polypeptide WO2001049861A1 (fr)

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CN 99127222 CN1301859A (zh) 1999-12-29 1999-12-29 一种新的多肽——人SpaseI酶13和编码这种多肽的多核苷酸

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0444759A1 (fr) * 1990-02-28 1991-09-04 Gist-Brocades N.V. Traitement de protéines
WO1998014461A1 (fr) * 1996-09-30 1998-04-09 Smithkline Beecham Plc NOUVEAUX spsB
EP0890641A1 (fr) * 1997-07-10 1999-01-13 Smithkline Beecham Corporation Nouvelle IspA
US5985638A (en) * 1997-03-26 1999-11-16 Incyte Pharmaceuticals, Inc. Microsomal signal peptidase subunit

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0444759A1 (fr) * 1990-02-28 1991-09-04 Gist-Brocades N.V. Traitement de protéines
WO1998014461A1 (fr) * 1996-09-30 1998-04-09 Smithkline Beecham Plc NOUVEAUX spsB
WO1998014563A1 (fr) * 1996-09-30 1998-04-09 Smithkline Beecham Plc POLYNUCLEOTIDES ET POLYPEPTIDES DE spsA
US5985638A (en) * 1997-03-26 1999-11-16 Incyte Pharmaceuticals, Inc. Microsomal signal peptidase subunit
EP0890641A1 (fr) * 1997-07-10 1999-01-13 Smithkline Beecham Corporation Nouvelle IspA

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