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WO2002053590A1 - Nouvelle enzyme fibrinolytique, polynucleotide codant ce polypeptide et utilisation correspondante - Google Patents

Nouvelle enzyme fibrinolytique, polynucleotide codant ce polypeptide et utilisation correspondante Download PDF

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Publication number
WO2002053590A1
WO2002053590A1 PCT/CN2001/001631 CN0101631W WO02053590A1 WO 2002053590 A1 WO2002053590 A1 WO 2002053590A1 CN 0101631 W CN0101631 W CN 0101631W WO 02053590 A1 WO02053590 A1 WO 02053590A1
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Prior art keywords
protein
earthworm
fibrinolytic enzyme
plasmin
polynucleotide
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PCT/CN2001/001631
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English (en)
Chinese (zh)
Inventor
Yuancong Zhou
Xiaoyan Zhong
Hong Zhu
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Shanghai Institute Of Biochemistry, Chinese Academy Of Sciences
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Publication of WO2002053590A1 publication Critical patent/WO2002053590A1/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
    • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology and medicine. Specifically, the present invention relates to a novel polynucleotide encoding plasmin-earthworm plasmin Z and a protein encoded by the polynucleotide. The invention also relates to a method for preparing and using the polynucleotide and protein, and a pharmaceutical composition containing the plasmin. Background technique
  • Earthworm fibrinolytic enzymes are a class of enzymes that can hydrolyze fibrin (pro) from earthworms. It is found in the digestive tract of earthworms and has a molecular weight from 15,000 to 60,000 daltons. Earthworm fibrinolytic enzymes are not a single enzyme, but a collective term for multiple proteolytic enzymes with the same function.
  • earthworm fibrinolytic enzymes have dual functions. In addition to directly hydrolyzing fibrin, they can also activate plasminogen into fibrinolytic enzymes, thus indirectly hydrolyzing fibrin.
  • the results of in vitro pharmacological experiments indicate that, in addition to directly dissolving blood clots, earthworm fibrinolytic enzymes can stimulate the release of tPA from vascular endothelial cells, thereby enhancing fibrinolysis in vivo.
  • the results of in vivo and in vitro thrombosis model experiments and artificial thrombolysis experiments indicate that earthworm fibrinolytic enzyme has obvious antithrombotic and thrombolytic effects.
  • earthworm fibrinolytic enzyme Based on the characteristics of earthworm fibrinolytic enzyme, it has been made into oral capsules at home and abroad for clinical treatment of thromboembolic diseases. For example, medicines such as Longxin, lumbrokinase, Brock, Puenfu, and thrombolytic capsules are all made from earthworm fibrinolytic enzyme. However, the structure of earthworm fibrinolytic enzymes is still unknown, and the amino acid sequence or nucleotide sequence of any specific earthworm fibrinolytic enzyme has not been reported.
  • An object of the present invention is to provide a novel plasmin, earthworm plasmin, and fragments, analogs and derivatives thereof.
  • Another object of the invention is to provide polynucleotides encoding these proteins.
  • a novel isolated earthworm fibrinolytic enzyme Z protein is provided.
  • the protein is derived from earthworms and comprises: a protein having the amino acid sequence of SEQ ID NO: 2, or a conservative variant protein thereof, or its activity Fragment, or an active derivative thereof.
  • the protein has SEQ ID NO: 2 Amino acid sequence of protein.
  • a polynucleotide encoding the isolated proteins, the polynucleotide comprising a nucleotide sequence having at least 70 nucleotides with a nucleotide sequence selected from the group consisting of % Homology: (a) a polynucleotide encoding the earthworm plasmin Z protein; and (b) a polynucleotide complementary to the polynucleotide (a).
  • the polynucleotide encodes a protein having the amino acid sequence shown in SEQ ID NO: 2.
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 1 to 723 in SEQ ID NO: 1; (b) a sequence having positions 1 to 979 in SEQ ID NO: 1 the sequence of.
  • a method for preparing a protein having earthworm fibrinolytic enzyme z protein activity comprising: (a) culturing the above-mentioned transformed or transduced under conditions suitable for expressing earthworm fibrinolytic enzyme Z Host cells; (b) isolating a protein with earthworm fibrinolytic Z protein activity from the culture.
  • an antibody that specifically binds to the earthworm fibrinolytic enzyme Z protein is provided.
  • a pharmaceutical composition which contains a safe and effective amount of the earthworm plasmin Z of the present invention and a pharmaceutically acceptable carrier.
  • These pharmaceutical compositions can treat conditions such as thromboembolic diseases.
  • Figure 1 shows the PCR amplification bands of earthworm fibrinolytic enzyme Z.
  • Lane 1 is the DNA standard;
  • lane 2 is the PCR amplification product.
  • Figure 2 shows the gene sequence of earthworm fibrinolytic enzyme Z and its encoded protein sequence.
  • Figure 3 shows the construction process of the prokaryotic expression vector pET-28a (+) expressing earthworm fibrinolytic enzyme Z.
  • Figure 4 shows the SDS-PAGE and western maps of the earthworm fibrinolytic Z expression products. The lanes are: 1. Standard molecular weight of protein, 2. Total protein of bacteria before IPTG induction, 3. Total protein of bacteria after IPTG induction, 4. Western map. detailed description
  • the inventors first isolated an earthworm fibrinolysis with a molecular weight of 25125. 0 Daltons from a variety of earthworms. Enzyme (mass spectrometry results), named it earthworm fibrinolytic enzyme Z. And, with William ’s cavity g uillelini) ⁇ , the amino acid sequence at the N-terminus of earthworm fibrinolytic enzyme Z was determined, and the corresponding primers were synthesized based on this. The relevant mRNA was isolated from the earthworm as a template, and cDNA was obtained by reverse transcription. The gene of earthworm fibrinolytic enzyme Z was amplified and cloned, and the full sequence of the gene was determined.
  • earthworm fibrinolytic enzyme Z was then inserted into a suitable vector and transferred into a suitable host cell, and earthworm fibrinolytic enzyme Z was expressed and isolated. And identified by Western blot analysis.
  • the terms "earthworm plasmin Z”, “earthworm plasmin Z protein”, or “earthworm plasmin Z polypeptide” are used interchangeably, and all refer to having an earthworm plasmin Z amino acid sequence (SEQ ID NO: 2). They include plasmin earthworm plasmin Z with or without the starting methionine. These terms also include earthworm plasmin Z with or without a signal peptide.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • the polynucleotide and protein in the natural state in a living cell are not separated and purified, but the same polynucleotide or protein is separated and purified if separated from other substances existing in the natural state. .
  • isolated earthworm plasmin Z polypeptide or protein means that earthworm plasmin Z protein is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated.
  • Those skilled in the art can isolate and purify the earthworm fibrinolytic enzyme ⁇ using standard protein purification techniques, especially FPLC.
  • the protein of the present invention may be a recombinant protein, a natural protein, or a synthetic protein, and preferably a recombinant protein.
  • the protein of the present invention may be a naturally purified product or a chemically synthesized product or produced from a prokaryotic or eukaryotic host (for example, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant technology.
  • a prokaryotic or eukaryotic host for example, bacteria, yeast, higher plants, insects, and mammalian cells
  • the protein of the invention may be glycosylated, or it may be non-glycosylated.
  • the proteins of the invention may also or may not include the initial methionine residue.
  • the invention also includes fragments, derivatives and analogs of earthworm fibrinolytic enzyme Z.
  • fragment refers to a protein that substantially retains the same biological function or activity of the natural earthworm fibrinolytic enzyme Z of the present invention.
  • the protein fragment, derivative or analog of the present invention may be a protein in which ⁇ has one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may be It may not be encoded by the genetic code, or (ii) a protein with a substituent group in one or more amino acid residues, or (iii) a mature protein with another compound (such as a compound that extends the half-life of a protein, such as polyethylene Diol), a protein formed by fusion, or (iv) a protein formed by fusing an additional amino acid sequence to this protein sequence (such as a leader sequence or a secreted sequence or a sequence used to purify this protein or a prion sequence, or an antigen IgG Fragment of the fusion protein).
  • the term "earthworm plasmin Z protein” refers to a protein of SEQ ID NO. 2 sequence having earthworm plasmin Z activity.
  • the term also includes variants of the sequence of SEQ ID NO. 2 having the same function as earthworm fibrinolytic enzyme Z. These variants include (but are not limited to): several (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid deletions, insertions And / or substitutions, and adding one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus.
  • substitution of amino acids with similar or similar properties usually does not change the function of the protein.
  • adding one or more amino acids to the C-terminus and / or N-terminus usually does not change the function of the protein.
  • the term also includes active fragments and active derivatives of earthworm fibrinolytic enzyme Z.
  • Variations of this protein include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA sites that can hybridize with earthworm plasmin Z DNA under high or low stringency conditions Encoded protein, and a polypeptide or protein obtained using an antiserum against earthworm plasmin Z protein.
  • the present invention also provides other proteins, such as a fusion protein comprising an earthworm fibrinolytic enzyme Z protein or a fragment thereof. In addition to the almost full-length protein, the invention also includes soluble fragments of the earthworm fibrinolytic enzyme Z protein sequence.
  • the fragment has at least about 10 consecutive amino acids, usually at least about 30 consecutive amino acids, preferably at least about 50 consecutive amino acids, and more preferably at least about 80 consecutive amino acids, and most preferably at least about 80 consecutive amino acids. To at least about 100 consecutive amino acids.
  • the invention also provides earthworm plasmin Z or an analogue.
  • the differences between these analogs and the natural earthworm fibrinolytic enzyme Z protein may be differences in amino acid sequences, differences in modified forms that do not affect the sequence, or both.
  • These proteins include natural or induced genetic variants. Induced variants can be obtained by a variety of techniques, such as random mutagenesis by radiation or exposure to mutagens, or by site-directed mutagenesis or other known molecular biology techniques.
  • Analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (such as ⁇ , y-amino acids). It should be understood that the protein of the present invention is not limited to the representative proteins exemplified above.
  • Modified (usually unchanged primary structure) forms include chemically derived forms of proteins in vivo or in vitro such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modification in the synthesis and processing of proteins or in further processing steps. This modification can be accomplished by exposing the protein to an enzyme that undergoes glycosylation, such as mammalian glycosylation or deglycosylation. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are proteins that have been modified to increase their resistance to proteolysis or to optimize their solubility.
  • glycosylation such as those produced by glycosylation modification in the synthesis and processing of proteins or in further processing steps. This modification can be accomplished by exposing the protein to an enzyme that undergoes glycosylation, such as mammalian glycosylation or deglycosylation. Modified forms also include sequences having
  • the "earthworm plasmin Z conservative variant protein” refers to the amino acid of SEQ ID NO: 2 Compared with sequences, there are at most 10, preferably at most 8 and more preferably at most 5 and most preferably at most 3 amino acids are replaced by amino acids with similar or similar properties to form a protein. These conservatively mutated proteins are preferably produced by amino acid substitution according to Table 1. Table 1
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature protein may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers in the present invention to a nucleic acid sequence that encodes a protein having SEQ ID NO: 2, but which differs from the coding region sequence shown in SEQ ID NO: 1.
  • the polynucleotide encoding the mature protein of SEQ ID NO: 2 includes: a coding sequence encoding only the mature protein; a coding sequence of the mature protein and various additional coding sequences; a coding sequence of the mature protein (and optional additional coding sequences); and Non-coding sequence.
  • the term "polynucleotide encoding a protein" may include a polynucleotide encoding the protein, or a polynucleotide that also includes additional coding and / or non-coding sequences.
  • the present invention also relates to variants of the above-mentioned polynucleotides, which encode fragments, analogs and derivatives of polypeptides or proteins having the same amino acid sequence as the present 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 without substantially altering the function of the protein it encodes .
  • the present invention also relates to a polynucleotide that hybridizes to the aforementioned sequence and has at least 50%, preferably at least 70%, and more preferably at least 80% homology between the two sequences.
  • the invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 X SSC, 0.1% SDS, 60 ° C ; or (2) during hybridization Added denaturing agents, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc .; or (3) the homology between only two sequences is at least 90% Above, and preferably above 95%, hybridization occurs.
  • the protein encoded by the hybridizable polynucleotide has the same biological function and activity as the mature protein shown in SEQ ID NO: 2.
  • nucleic acid fragment that hybridizes to the sequence described above.
  • a "nucleic acid fragment” has a length of at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more.
  • Nucleic acid fragments can be used in nucleic acid amplification techniques such as PCR to identify and / or isolate polynucleotides encoding earthworm plasmin Z.
  • the protein and polynucleotide in the present invention are preferably provided in an isolated form, and more preferably purified to homogeneity.
  • the full-length Z-nucleotide sequence of the earthworm fibrinolytic enzyme of the present invention or a fragment thereof can usually be obtained by a PCR amplification method, a recombinant method, or a synthetic method.
  • primers can be designed according to the disclosed nucleotide sequence ⁇ I, especially the open reading frame sequence, and a commercially available cDNA library or a conventional method known to those skilled in the art can be used.
  • the prepared cDNA library was used as a template and amplified to obtain the relevant sequences.
  • Relevant sequences can also be obtained directly by RT-PCR. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then stitch the amplified fragments together in the correct order.
  • the recombination method can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • relevant methods can also be synthesized by artificial synthesis, especially when the fragment length is short. Pass Often, long fragments can be obtained by first synthesizing multiple small fragments and then performing ligation.
  • a DNA sequence encoding a protein (or a fragment thereof, or a derivative thereof) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into a variety of existing DNA molecules (or such as vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • a method for amplifying DNA / RNA using PCR technology is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-cDNA terminal rapid amplification method
  • the primers used for PCR may be appropriately selected based on the sequence information of the present invention disclosed herein. And can be synthesized by conventional methods.
  • the amplified DNA / RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.
  • 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 earthworm plasmin Z coding sequence, and a method for producing the protein of the present invention by recombinant technology.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant earthworm plasmin Z protein. Generally there are the following steps:
  • the earthworm plasmin Z polynucleotide sequence can be inserted into a recombinant expression vector.
  • recombinant expression 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-based expression vectors expressed in bacteria; pMSXND expression vectors expressed in mammalian cells; and baculovirus-derived vectors expressed in insect cells.
  • any plasmid and vector can be used as long as it can be replicated and stabilized in the host.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translation control elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing earthworm fibrinolytic enzyme Z-encoding DNA sequences and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombinant technology.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • 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 vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express a protein.
  • the host cell can be 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.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: E. coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or S 9; CHO, COS, 293 cells, or Bowes melanoma cells Animal cells and so on.
  • Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, that 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.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of DNA uptake can be harvested after exponential growth phase, with (& (: Treatment 1 2, steps well known in the art using another method is to use MgCl. 2.
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, and lipid Body packaging, etc.
  • the obtained transformants can be cultured by a conventional method to express a protein encoded by the gene of the present invention.
  • 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 protein in the above method may be expressed intracellularly, or on a cell membrane, or secreted extracellularly. 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. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic disruption, ultra-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.
  • Recombinant earthworm plasmin Z or protein has many uses. These uses include (but are not limited to): direct use as a drug to treat thromboembolic diseases, and to screen antibodies, proteins, or other ligands that promote or combat earthworm fibrinolytic enzyme Z function. Screening the protein library with the expressed recombinant earthworm fibrinolytic enzyme Z can be used to find therapeutic protein molecules that can inhibit or stimulate the function of earthworm fibrinolytic enzyme Z.
  • the present invention also includes polyclonal antibodies and monoclonal antibodies, especially monoclonal antibodies, having specificity for the protein encoded by earthworm fibrinolytic enzyme Z DNA or fragments thereof.
  • specificity means that the antibody is capable of binding to the earthworm fibrinolytic enzyme Z gene product or fragment.
  • it refers to those antibodies that can bind to earthworm plasmin Z gene products or fragments but do not recognize and bind to other unrelated antigen molecules.
  • the antibodies in the present invention include those molecules capable of binding and inhibiting earthworm fibrinolytic enzyme z, as well as those which do not affect the function of earthworm fibrinolytic enzyme z.
  • the invention also includes those antibodies that are capable of binding to a modified or unmodified form of the earthworm plasmin Z gene product.
  • the invention includes not only intact monoclonal or polyclonal antibodies, but also antibody fragments with immunological activity, such as Fab 'or (Fab) 2 fragments; antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules; Or chimeric antibodies, such as antibodies that have murine antibody binding specificity but still retain the antibody portion from humans.
  • Fab Fab 'or
  • the antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, a purified earthworm plasmin Z gene product or an antigenic fragment thereof can be administered to an animal to induce the production of polyclonal antibodies. Similarly, cells expressing earthworm plasmin Z or its antigenic fragments can be used to immunize animals to produce antibodies.
  • the antibody of the present invention may be a monoclonal antibody. Such monoclonal antibodies can be prepared using hybridoma technology.
  • the various antibodies of the present invention can be obtained by conventional immunological techniques using fragments or functional regions of the earthworm fibrinolytic enzyme z gene product. These fragments or functional regions can be prepared by recombinant methods or synthesized using a protein synthesizer.
  • Antibodies that bind to the unmodified form of the earthworm fibrinolytic enzyme z gene product can be produced by immunizing animals with gene products produced in prokaryotic cells (for example, Co //); Phosphorylated proteins or polypeptides) can be obtained by immunizing animals with gene products produced in eukaryotic cells, such as yeast or insect cells.
  • prokaryotic cells for example, Co //
  • Phosphorylated proteins or polypeptides can be obtained by immunizing animals with gene products produced in eukaryotic cells, such as yeast or insect cells.
  • earthworm fibrinolytic enzyme Z of the present invention through various conventional screening methods, substances that interact with the earthworm fibrinolytic enzyme Z, such as receptors, inhibitors, agonists or antagonists, can be screened.
  • the earthworm fibrinolytic enzyme Z, and the antibody, inhibitor, agonist, or antagonist of the earthworm of the present invention, when administered therapeutically (administration), can provide different effects.
  • these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5 to 8, preferably pH 6 to 8, although the pH can be varied with The nature of the formulation and the condition to be treated will vary.
  • Formulated drug group The compounds can be administered by conventional routes including, but not limited to: intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or local administration.
  • the protein of the present invention can be directly used for the treatment of diseases, for example, for the treatment of thrombosis.
  • other therapeutic agents such as antitumor drugs, can also be used simultaneously.
  • the invention also provides a pharmaceutical composition containing a safe and effective amount of the earthworm fibrinolytic enzyme Z protein of the invention and a pharmaceutically acceptable carrier or excipient.
  • Such carriers include (but are not limited to): saline, buffers, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods.
  • Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example, from about 0.1 micrograms per kilogram body weight to about 5 milligrams per kilogram body weight per day.
  • the protein of the present invention can also be used with other therapeutic agents.
  • a safe and effective amount of earthworm fibrinolytic Z is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms / day, and in most cases not more than about 8 mg / kg body weight, Preferably the dose is from about 10 micrograms / day to about 1 mg / kg body weight.
  • the specific dosage should also consider factors such as the route of administration, the patient's health, etc., which are all within the skill of a skilled physician.
  • a method for detecting the presence or absence of earthworm plasmin Z in a sample is to use a specific antibody for earthworm plasmin Z, which comprises: contacting the sample with earthworm plasmin z specific antibody; and observing whether an antibody complex is formed
  • the formation of an antibody complex indicates the presence of earthworm fibrinolytic enzyme z in the sample.
  • the advantage of the present invention is that the earthworm contains more than 10 kinds of plasmin. It is difficult to separate and purify one of the components from earthworms to a single purity.
  • the earthworm fibrinolytic enzyme z gene was cloned, its gene sequence was analyzed and determined, and it was successfully expressed.
  • the earthworm fibrinolytic enzyme Z of the present invention provides a new therapeutic approach for the treatment of diseases such as thromboembolic diseases, and thus has great application prospects.
  • the present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention.
  • the experimental methods without specific conditions in the following examples are usually performed according to the conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer Suggested conditions.
  • Example 1 Example 1
  • peaks are isozymes of earthworm fibrinolytic enzyme, and the third peak has The most significant plasmin activity. It was dissolved in distilled water and separated and purified through multiple steps such as an ion exchange column, an affinity chromatography column, and a molecular sieve column to obtain a purified sample of earthworm fibrinolytic enzyme. The samples were analyzed by HPLC or SDS electrophoresis and showed a single band. Then, the molecular weight of the earthworm fibrinolytic enzyme was measured by mass spectrometry, and the result was 25125. 0 Dalton. The enzyme is named earthworm fibrinolytic enzyme Z. The four earthworms tested contained this earthworm fibrinolytic enzyme Z, which indicates that earthworm fibrinolytic enzyme Z is a ubiquitous fibrinolytic enzyme.
  • Example 2
  • the upstream oligonucleotide primer was designed as: 5'ATGAATCCATGATC C TGGGAG (T) GG (ATC) ACG (ACT) A (GC) A3 '(SEQ ID NO:
  • the supernatant was aspirated into a new centrifuge tube, and 0.4 mL of 2 mol / L NaAc, Ph 4.0, 4 mL of water-saturated phenol and 2 mL of chloroform: isoamyl alcohol (49: 1) were added. After shaking, the solution was ice-baked for 15 min. Centrifuge at 10,000g at 4 ° C for 20min. Take the upper aqueous phase and mix with an equal volume of isopropanol, and leave it at -20 ° C for 1 h. 4 ° C, 10,000 g centrifugation for 20 minutes to collect the precipitate.
  • the precipitate was re-dissolved in 2 mL of solution D, and incubated for 2 to 3 rain, to make it fully dissolved, 0.2 mL 2 mol / L NaAc, pH 4.0 and 2 mL isopropyl Alcohol, mix for 1 h at -20 ° C. 4 ° ⁇ , 10, 00 (centrifugation 2 (1 ⁇ 2), the precipitate was washed with 75% ethanol, and left open in the air for several minutes to fully evaporate the ethanol. Finally dissolved in DEPC (diethyl pyrocarbonate) treatment Take 30 ⁇ of sterile water. Take 1 ⁇ to measure the RNA's 0D 26 / 0D 28. , ⁇ ⁇ take formaldehyde denaturing electrophoresis to determine the purity and integrity of the RNA.
  • DEPC diethyl pyrocarbonate
  • RNA of William's columbaria vermicularis prepared in Example 4 was used as a template, and 3'RACE KIT was used for reverse transcription by Bowringman Company. The operation was performed according to the instructions.
  • the primers synthesized in Example 3 were used for PCR amplification from the cDNA obtained above as a template.
  • the total reaction system was 50 ⁇ , where the final concentration of MgCl 2 was 1.5 m mol / L, and the final concentration of dNTP was 20 ⁇ mol / L.
  • the concentration of the primer was 30 n mol / L, and the unit of Tagase 2 (purchased from Gibco BRL) ).
  • the reaction conditions are: denaturation at 94 ° C for 3 min, and then entering the cycle.
  • the amplified nucleic acid is an unknown sequence, in order to avoid the occurrence of coincident restriction sites inside the gene, which may cause the gene to be cleaved and destroy its integrity, a T-vector was used for cloning.
  • T-carrier the vector pBluescript-SK (purchased from Stratagene) was cut with ⁇ : oRI, equal volume of phenol: chloroform treatment, ethanol precipitation, dissolved in 50 ⁇ system, containing lOxPCR buffer 5 ⁇ , lOOra mol / L ⁇ 2 ⁇ , Taq enzyme 1 unit. Incubate at 70 ° C for 2h, treat with an equal volume of phenol: chloroform. After ethanol precipitation, dissolve in 10 ⁇ l sterilized water.
  • a male New Zealand big-eared rabbit weighing about 2 kg was taken.
  • the 1mg earthworm plasmin Z sample was ground into latex with Freund's complete adjuvant, and injected at multiple points in the rabbit's core. After half a month's rearing, the sample was ground into latex with lmg earthworm plasmin Z sample and Freund's incomplete adjuvant, and then injected into the rabbit's core at multiple points.
  • 1.5 mg earthworm plasmin Z sample plus Freund's incomplete adjuvant was used to boost immunity in the same way.
  • Half a month later another 1 mg of earthworm plasmin Z plus Freund's incomplete adjuvant was used to boost the immunity again. After half a month of breeding, blood was collected from the arterioles, left at 4 ° C overnight, and centrifuged at 2,000 rpm for 3 minutes. The upper serum is rabbit anti-earthworm plasmin Z antibody.
  • the positive clones obtained in Example 6 were entrusted to Shanghai Ji Kang Biotechnology Company for determination.
  • the complete gene sequence and the encoded protein sequence are shown in Figure 2.
  • the gene is shown in SEQ ID NO: 1, and has a length of 924 bp, with a reading frame length of 723 bp (positions 1-723), and encodes 241 amino acids (SEQ ID NO: 2).
  • the 3′-untranslated region after 723 bp has two polyA tail signals AATAAA.
  • the earthworm plasmin Z cDNA encodes a protein sequence that is 54% homologous to a protein encoded by another cDNA called Uwibricus bi mast us. Therefore, this is a new unreported earthworm plasmin gene. From the amino acid sequence encoded by this new gene, the molecular weight calculated by computer software DNASTAR is 25138. 10, which is consistent with the molecular weight of plasmin Z extracted from earthworms.
  • the translation region of the gene was expressed in the prokaryotic expression vector pET-28a (+) (Novagen), and was prepared in Example 7 Western blot analysis of rabbit anti-earthworm fibrinolytic enzyme ⁇ antibody and expression products.
  • the plasmid construction map is shown in Figure 3, and the expression results are shown in Figure 4.
  • the upstream primer was 5'GCGAATTCATCCTGGGAGGGACGAAA3 '(primer 3) (SEQ ID NO: 4), and the downstream primer Is: 5'CACTCGAGTTAGTGCAGTCGGCTCCA3 '(primer 4) (SEQ ID NO: 5).
  • the full-length cDNA was used as a template, and PCR amplification was performed as described above.
  • the amplified product fcoRlAttoI was digested and cloned into the 0 RlA1 ⁇ 2oI site of pET-28a (+).

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Abstract

Cette invention se rapporte à un nouveau type d'enzyme fibrinolytique, l'enzyme fibrinolytique Z provenant du lombric, à un polynucléotide codant cette enzyme fibrinolytique Z et à un procédé permettant de produire cette enzyme fibrinolytique Z par technique de recombinaison. Cette invention concerne également un procédé destiné à utiliser cette enzyme fibrinolytique Z dans le traitement de divers types de maladies, telles que les maladies thromboemboliques. Une composition de médicament contenant cette enzyme fibrinolytique Z dérivée du lombric est également décrite.
PCT/CN2001/001631 2000-12-29 2001-12-14 Nouvelle enzyme fibrinolytique, polynucleotide codant ce polypeptide et utilisation correspondante WO2002053590A1 (fr)

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CN100355884C (zh) * 2005-01-12 2007-12-19 华东理工大学 一种蚯蚓纤溶酶及其编码序列与用途
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XIONG YI ET AL.: "Purification and determination of partial sequence of earthworm fibrinolytic enzyme", CHINESE BIOCHEMICAL JOURNAL, vol. 13, no. 3, June 1997 (1997-06-01), pages 292 - 296 *
ZHAO XIAOYU ET AL.: "Component analysis of fibrinolytic enzyme from earthworm", CHINESE JOURNAL OF BIOCHEMISTRY AND MOLECULAR BIOLOGY, vol. 14, no. 4, August 1998 (1998-08-01), pages 407 - 411 *

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