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WO2001074867A1 - Nouveau polypeptide, proteine humaine 11 regulant la protease, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, proteine humaine 11 regulant la protease, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001074867A1
WO2001074867A1 PCT/CN2001/000200 CN0100200W WO0174867A1 WO 2001074867 A1 WO2001074867 A1 WO 2001074867A1 CN 0100200 W CN0100200 W CN 0100200W WO 0174867 A1 WO0174867 A1 WO 0174867A1
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Prior art keywords
polypeptide
polynucleotide
proteolytic enzyme
protein
human proteolytic
Prior art date
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PCT/CN2001/000200
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biowindow Gene Development Inc. Shanghai filed Critical Biowindow Gene Development Inc. Shanghai
Priority to AU44045/01A priority Critical patent/AU4404501A/en
Publication of WO2001074867A1 publication Critical patent/WO2001074867A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • E. coli can be infected by obligate parasites, a double-stranded DNA virus, lambda phage.
  • Lambda phage is a mild phage that can proliferate in two states in E. coli. One is in a lysed state, and phage DM autonomously replicates and directs the formation of capsid proteins, packaging, and host cell lysogens. The other is a lysing state, where lambda MA is integrated into the host cell genome and replicated.
  • Bacteriophages are called phages; hosts are called lysosomes and are considered immune.
  • the lambda ⁇ protein increases the lysogenicity of various lambda phages that express different C II proteins.
  • the excessive production of CIII may induce a heat shock response by fixing an RNA polymerase, a heat shock specific subunit of ⁇ 32 .
  • cm directly inhibits proteolytic enzymes by binding to stabilized proteins or by binding to specific cellular proteolytic enzymes of these proteins.
  • the central part of the cm protein in different lambda phages is the region with the most similar sequence, and the activity of this protein is necessary and sufficient.
  • the human proteolytic enzyme regulatory protein 11 protein plays an important role in regulating important functions of the body such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, so there has been a need in the art to identify more involved in these During the process of human proteolytic regulator 11 protein, the amino acid sequence of this protein was specifically identified.
  • the separation of the new human proteolytic enzyme regulatory protein 11 protein also provides the basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic agents for the disease, so it is important to isolate its coding DNA. Disclosure of invention
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human protein hydrolysate regulating protein 11.
  • Another object of the present invention is to provide a method for producing human proteolytic enzyme-modulating protein 11.
  • Another object of the present invention is to provide a human proteolytic enzyme regulating protein directed to 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 in human proteolytic enzyme regulating protein 11.
  • the present invention relates to an isolated polypeptide, which is of human origin, and includes: 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:
  • 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 present invention also relates to a method for detecting a disease or disease susceptibility related to abnormal expression of human proteolytic regulator 11 protein in vitro, which comprises detecting a mutation in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, or detecting The amount or biological activity of a polypeptide of the invention in a biological sample.
  • Regular refers to a change in the function of human proteolytic enzyme regulatory protein 11, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological, functional, or immune properties of human proteolytic enzyme regulatory protein 11. change.
  • substantially pure is meant substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify human proteolytic enzyme regulatory protein 11 using standard protein purification techniques. Basically pure human proteolytic enzyme regulatory protein 11 produces a single main band on a non-reducing polyacrylamide gel. The purity of the human proteolytic enzyme 11 can be analyzed by amino acid sequence analysis.
  • “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. The inhibition of such hybridization can be detected by performing hybridization (Southern blotting 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 strict Conditions with reduced sex allow non-specific binding because conditions with 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 software package, DNASTAR, Inc., Madi son Wis.). The MEGAUGN program can compare two or more sequences according to different methods such as the Clus ter method (Higg ins, DG and PM Sharp (1988) Gene 73: 237-244). 0 The Cluster method checks each pair by checking the distance between all pairs. Group sequences are arranged in clusters. The clusters are then assigned in pairs or groups.
  • '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 emzumology 183: 625-645) 0 "similarity" means The degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • isolated human proteolytic enzyme regulatory protein 11 means that human proteolytic enzyme regulatory protein 11 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 proteolytic enzyme regulatory protein 11 using standard protein purification techniques. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. Human proteolytic enzymes can regulate the purity of protein 11 polypeptide by amino acid sequence analysis.
  • the invention also includes fragments, derivatives and analogs of human proteolytic enzyme regulatory protein 11.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human proteolytic enzyme regulating protein 11 of 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.
  • the present invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50% identity between the two sequences, preferably 703 ⁇ 4 identity).
  • the invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions.
  • 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 proteolytic enzyme regulatory protein 11 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • Amplification of DM / RNA by PCR (Saiki, et al. Science 1985; 230: 1350-1354) are preferred for obtaining the genes of the 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 DNA fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, the sequencing must be repeated. Sometimes the cMA sequences of multiple clones need to be determined in order to splice into full-length cDM sequences.
  • a polynucleotide sequence encoding a human proteolytic enzyme regulatory protein 11 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 a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If desired, 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.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated human proteolytic enzyme regulatory protein 11.
  • llkDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Pr imerl 5'- GTTCTTCTTCTGCATTAAGGATGT -3 '(SEQ ID NO: 3)
  • Pr imerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Pr imer4 5 '-CATGGATCCTTAAAAAAATTCCCCTTCTACAGA- 3' (Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and BamHI digestion sites, respectively, followed by the coding sequences of the 5' and 3 'ends of the target gene, respectively.
  • the Ndel and BamHI restriction sites correspond to the selective endonuclease sites on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865. 3).
  • the pBS-0159E12 plasmid containing the full-length target gene was used as a template for the PCR reaction.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthetic polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature), so that the hybridization background is reduced and only strong specific signals are retained.
  • NC membrane nitrocellulose membrane
  • Gene chip or gene microarray is a new technology that many national laboratories and large pharmaceutical companies are currently researching and developing. It refers to the orderly and high-density arrangement of a large number of target gene fragments on slopes. , Silicon and other carriers, and then use fluorescence detection and computer software to compare and analyze the data, in order to achieve the purpose of rapid, efficient, high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as a target DM 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 genetic diseases . The specific method steps have been reported in the literature.
  • the phage regulatory protein cm directly inhibits proteolytic enzymes by binding to stabilized proteins or by binding to specific cellular proteolytic enzymes of these proteins. Excessive production of CIII may induce a heat shock response by fixing an RNA polymerase, a heat shock specific subunit of ⁇ 32 .
  • CIII itself may be a gene expression regulator, and the conserved helix domain of cm is considered to be an active site for regulating proteolytic enzymes and degrading C II and ⁇ 32 proteins.
  • the membrane preparation of 11 was cultured with labeled human proteolytic enzyme regulating protein 11. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human proteolytic enzyme regulatory protein 11 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human proteolytic regulator 11 can bind to human proteolytic regulator 11 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 exert its biology Features.
  • Antibodies against human proteolytic enzyme 11 can be used in immunohistochemistry to detect human proteolytic enzyme 11 in biopsy specimens.
  • Antibodies can also be used to design immunotoxins against a specific bead site in the body.
  • human proteolytic enzyme-modulated protein 11 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 proteolytic enzyme regulating protein 11 positive cell.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human proteolytic enzyme regulating protein 11.
  • Administration of appropriate doses of antibodies can stimulate or block the production or activity of human proteolytic enzyme-regulated protein 11.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human proteolytic enzyme regulating protein 11.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human proteolytic regulator 11 detected in the test can be used to explain human proteolytic regulator 11 Importance in various diseases and for diagnosing diseases in which human proteolytic regulator 11 plays a role.
  • the polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzyme, and can be analyzed by one-dimensional or two-dimensional or three-dimensional gel electrophoresis, and more preferably by mass spectrometry coding.
  • PCR localization of somatic hybrid cells is a quick way to localize DM to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cMA libraries.
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendel ian Inher i tance in Man (available through contact with Johns Hopkins Univers i ty Welch Medical Library available online). Linkage analysis can then be used to determine the relationship between genes and diseases that are mapped to chromosomal regions.
  • 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 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 proteolytic enzyme 11 is administered in an amount effective to treat. And / or prevent a specific indication.
  • the amount and range of human proteolytic regulator 11 to be administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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  • Gastroenterology & Hepatology (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract

L'invention concerne un nouveau polypeptide, une protéine humaine 11 régulant la protéase, 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, des troubles du développement de l'embryon et des troubles de la croissance et du développement. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la protéine humaine 11 régulant la protéase.
PCT/CN2001/000200 2000-03-07 2001-02-26 Nouveau polypeptide, proteine humaine 11 regulant la protease, et polynucleotide codant pour ce polypeptide WO2001074867A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU44045/01A AU4404501A (en) 2000-03-07 2001-02-26 A novel polypeptide-human proteolytic enzyme regulatory protein 11 and the polynucleotide encoding said polypeptide

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN00111897A CN1312261A (zh) 2000-03-07 2000-03-07 一种新的多肽——人蛋白水解酶调节蛋白11和编码这种多肽的多核苷酸
CN00111897.8 2000-03-07

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5599906A (en) * 1990-04-13 1997-02-04 Schering Corporation Protease assays

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5599906A (en) * 1990-04-13 1997-02-04 Schering Corporation Protease assays
US5721133A (en) * 1990-04-13 1998-02-24 Schering Corporation Protease assays

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AU4404501A (en) 2001-10-15

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