WO2005100570A1 - Gene enzymatique fibrinolytique de lombric, souches de genie genetique, et leur construction et leurs utilisations - Google Patents

Gene enzymatique fibrinolytique de lombric, souches de genie genetique, et leur construction et leurs utilisations Download PDF

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Publication number
WO2005100570A1
WO2005100570A1 PCT/CN2005/000501 CN2005000501W WO2005100570A1 WO 2005100570 A1 WO2005100570 A1 WO 2005100570A1 CN 2005000501 W CN2005000501 W CN 2005000501W WO 2005100570 A1 WO2005100570 A1 WO 2005100570A1
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plasmin
genetically engineered
protein
gene
pichia pastoris
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PCT/CN2005/000501
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English (en)
Chinese (zh)
Inventor
Xiaolin Meng
Jinping Xu
Yan Hu
Junjie Zhang
Wei Lu
Jian Wang
Haiyang Meng
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Green Life Laboratory Limited
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Priority claimed from CN 200410034475 external-priority patent/CN1563387A/zh
Priority claimed from CN 200410034474 external-priority patent/CN1259411C/zh
Application filed by Green Life Laboratory Limited filed Critical Green Life Laboratory Limited
Publication of WO2005100570A1 publication Critical patent/WO2005100570A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6435—Plasmin (3.4.21.7), i.e. fibrinolysin
    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21007—Plasmin (3.4.21.7), i.e. fibrinolysin

Definitions

  • the present invention relates to a nucleotide sequence of the genus ⁇ ⁇ plasmin gene of the genus Orthopaedic genus and the gene encoding the karst plasmin gene of the genus Orthodonta.
  • the invention also relates to a method for preparing a sputum plasmin gene of the genus Zhengsui.
  • the invention further relates to the construction of a Pichia pastoris genetically engineered strain expressing the gene and the use of genetic engineering plasmin in drug research and development.
  • Cardiovascular and cerebrovascular diseases such as acute myocardial infarction, cerebral infarction, pulmonary embolism, peripheral venous thrombosis and deep venous thrombosis are one of the major diseases that endanger human life and health. According to the World Health Organization, about 12 million people worldwide die from heart disease and stroke each year. In China, with the rapid development of the economy and the improvement of the people's material living standards, the incidence of cardiovascular and cerebrovascular diseases and their mortality rate are also increasing year by year.
  • the pharmacological mechanisms of Thousand Dissolved Thrombolytic Therapy mainly include: 1) It has a special affinity with fibrin, not only hydrolyzes fibrinogen rich in plasminogen, but also hydrolyzes fibrin without plasminogen, which rapidly degrades fibrin. It also hydrolyzes fibrinogen. 2) Similar to tPA, it activates plasminogen and exerts an indirect effect. In vitro, it indicates that plasmin has a stimulating effect on the release of tPA in bovine vascular endothelial cells. 3) Hydrolysis of factor I inhibits the adhesion of platelets on its surface.
  • plasmin in sputum has multiple components, and at least 6 different components have been isolated only in Lumbricus mbellus.
  • Six cDNA sequences encoding plasmin genes have been isolated from the genus Hymenoptera.
  • the sputum fibrinolytic enzyme gene has also been cloned from the scorpion, such as Eisenia foetida, and the double-breasted scorpion, but there is no report that the plasmin gene was isolated and cloned and expressed.
  • sputum fibrinolytic enzyme preparations have been used in clinical practice, mainly for various capsule preparations, which are extracted from the intestinal cavity and body fluids, such as Puenfu, Baiao, Boroc, and thrombolytic capsules. More used in cardiovascular and cerebrovascular diseases.
  • fibrinase can improve blood rheology and reduce platelet aggregation rate, it can be used in some diseases related to blood rheology, such as Meniere's disease, sudden ear, and pulmonary heart disease.
  • Traditional sputum fibrinolytic enzyme capsule preparation has certain curative effect in clinical application, but it also has obvious defects.
  • the composition is complex, it can not be injected intravenously, and oral administration is slow, so it can not be used to treat acute heart and brain blood vessels. Embolism disease;
  • the production process of plastic capsule is complicated, the production is limited by the season and cycle of the cultivation, the product yield and quality are unstable; the process of separating and purifying plasmin from the worm is cumbersome, and the product may contain various small molecular peptides or Other chemicals may cause an allergic reaction in the body.
  • the genetic engineering method can obtain a large amount of fibrinolytic enzyme by simple fermentation, and the separation and purification process is simple, the product purity is high, and the cost is low.
  • genetically engineered plasmin has a wide range of uses in the research and development of medical, health and health care drugs, and can be used for the preparation of injectables, oral preparations, etc. for the treatment of embolic cardiovascular diseases, and can also be used for production prevention.
  • Thrombosis disease health care products that are beneficial to cardiovascular health.
  • the E. coli expression system is the most widely used and mature expression system. It is suitable for the expression of non-glycosylated proteins and proteins with simple secondary structure.
  • the expression products generally form inclusion bodies in the cytoplasm, and the products need to be renatured. It has activity, and some carriers can secrete the protein of interest in the intercellular substance or soluble in the cytoplasm.
  • the biggest drawback is the inability to perform post-expression processing and modification (such as glycosylation), which is required for many proteins to acquire function.
  • CHO cell expression system This is an expression system with relatively large construction and expression difficulty. It is suitable for expressing glycosylated proteins and proteins with complex spatial structure.
  • the expression products are generally present in the cytoplasm, if the gene is constructed before the target protein. Adding a signal peptide, the expression product will be secreted extracellularly, and the cells will be packaged into active proteins after translation, but the expression is low, the cost is high, and the purification is difficult, making it difficult to apply in industrial production. Therefore, in the development and application of sputum fibrinolytic enzyme, an expression system with high expression, good stability, high activity, protein post-translation processing, and industrial production is needed.
  • One object of the present invention is to provide a genus Phytophthora genus fibrinolytic enzyme gene and a nucleotide sequence thereof SEQ ID NO: 1.
  • One of the objects of the present invention is to provide a glutinine plasmin protein encoded by the ⁇ ⁇ ⁇ ⁇ ⁇ plasmin gene.
  • the ⁇ ⁇ ⁇ plasmin protein comprises the amino acid sequence of SEQ ID NO: 2.
  • One of the objects of the present invention is to provide a method for preparing a sputum plasmin gene of the genus Orthodox.
  • One of the objects of the present invention is to provide a genetically engineered strain capable of expressing sputum fibrinolytic enzyme.
  • the invention also relates to a method for preparing the strain for preparing a genetically engineered plasmin protein.
  • One of the objects of the present invention is to provide a method for preparing and cloning Escherichia coli fibrinolytic enzyme gene into Escherichia coli (E. coli).
  • One of the objects of the present invention provides a method for constructing a Pichia pastoris strain, which can be used to express a sputum plasmin gene to obtain a genetically engineered plasmin protein.
  • One of the objects of the present invention is to provide a genetically engineered Pichia pastoris strain Pichia pastoris X-33 (Ppic6 a A-EFE).
  • Another object of the present invention is to provide a medicament for a disease associated with blood rheology, characterized in that the medicament comprises genetically engineered mound plasmin protein and a pharmaceutically acceptable carrier.
  • Another object of the present invention is to provide an agent for treating embolic cardiovascular and cerebrovascular diseases, characterized in that the agent comprises a genetically engineered ⁇ plasmin protein.
  • Another object of the present invention is to provide an agent for treating embolic cardiovascular and cerebrovascular diseases, characterized in that the agent comprises a genetically engineered ⁇ plasmin protein and a pharmaceutically acceptable carrier.
  • Another object of the present invention is to provide a health care product which is useful for preventing cardiovascular health of a thrombotic disease.
  • FIG. 1 shows the results of PCR amplification of the plasmin gene.
  • Lane 1 is the DNA molecular weight standard (DNA/Hindlll);
  • Lane 2 is the first strand of cDNA synthesized by reverse transcription;
  • Lane 3 is the PCR amplification product of the plasmin gene.
  • FIG. 2 Enzyme of recombinant plasmid pUCm-T-EFE Cut the identification results.
  • Lane 1 is the DNA molecular weight standard (DNA/ Hind III); PCR amplification products of lane 2, 3 lysozyme gene, lane 4 is the DNA molecular weight standard ( ⁇ DNA/EcoR 1+ Hind III );
  • lane 5 is the recombinant plasmid pUCm -T-EFE was digested with Not I and Sal I;
  • Lane 6 is the result of single digestion with the recombinant plasmid pUCm-T-EFE by Sal I;
  • Lane 7 is the recombinant plasmid pUCm-T-EFE with Sph I single enzyme The result of the cut.
  • Figure 3 shows the results of restriction enzyme digestion of recombinant expression vectors pPIC6 oc A-EFE and pPIC6A-EFE.
  • Lane 1 is the PCR molecular weight standard
  • Lane 2 is the PCR identification result of the recombinant expression vector
  • Lane 3 is the result of double digestion of the recombinant expression vector with EcoR I+Xba I
  • swimming i £ 4 is the recombinant expression vector and Sac I single digestion Results
  • Lane 5 is the DNA molecular weight standard (DNA/EcoR I+ Hind III ).
  • Figure 4 shows the results of SDS-PAGE electrophoresis after purification of genetically engineered plasmin.
  • Lane 1 is a small molecular weight protein standard
  • Lane 2 is a purified genetically engineered Pichia pastoris X-33 (pPIC6 a A-EFE) supernatant, a genetically engineered plasmin protein.
  • Figure 5 shows the biological activity of plasmin by fibrin plate method.
  • a is the purified genetically engineered plasmin protein
  • b is lOul plasmin standard (10000 U/ml)
  • c is the fermentation product of 10 ⁇ l of the control Pichia pastoris X-33 (pPIC6 ⁇ ⁇ ) supernatant purified.
  • Figure 6 Determination of the biological activity of plasmin by fibrin plate method a is genetically engineered plasmin protein fermentation supernatant; b is lOul plasmin standard (10000 U/ml); c is lOul control pichia pastoris X-33 (pPIC6A) fermentation product. DETAILED DESCRIPTION OF THE INVENTION Preferred embodiments of the invention are described below.
  • the invention provides a nucleotide sequence of a sputum sputum plasmin gene of Orthodontic genus and a preparation method thereof.
  • a nucleotide sequence of a scorpion scorpion plasmin gene and a preparation method thereof are provided, and the method is a genus of genus
  • a plasmin gene cDNA was obtained by a reverse transcription PCR method using a specific oligonucleotide primer.
  • the powder was purchased from Huazhong Agricultural University.
  • the plasmin protein of the present invention has a molecular weight of 26.4 kD, an isoelectric point of 4.5, and is rich in glycine, serine, proline and aspartic acid. Its biochemical properties are:
  • the plasmin gene according to the present invention can also be obtained by the following methods: (1) subcloning: using the gene fragment of the present invention as a template, or using a recombinant plasmid containing the gene fragment of the present invention as a template, or The genetically engineered strain DNA containing the gene fragment of the present invention is used as a template, and the corresponding gene fragments are subcloned by conventional molecular biological manipulation methods (such as PCR), and these fragments may include all or part of the gene sequences involved in the present invention.
  • fragments can be expressed in bacteria, yeast, animal and plant cells or other eukaryotic or prokaryotic organisms to obtain protein or polypeptide fragments which have the same biological functions as the proteins involved in the present invention;
  • Synthetic According to the amino acid or nucleotide sequence involved in the present invention, a DNA fragment can be synthesized by chemical synthesis, and cloned into a corresponding vector for expression, and the expressed protein or polypeptide has the same protein as the protein of the present invention. Biological function.
  • the gene fragment of the present invention may be modified, for example, to mutate certain nucleotide sequences of the gene, but does not affect the encoded amino acid sequence of the protein; the amino acid or nucleotide sequence involved in the present invention may be added or deleted. Modification does not affect the biochemical properties of proteins, advanced structures, and biological functions of proteins, such as: secretion of signal peptides at the N-terminus or C-terminus of a protein or peptide, plus appropriate linkers (eg, 6 Histidine) facilitates protein extraction and purification.
  • the gene involved in the present invention can be cloned into a corresponding host after ligation to the vector to facilitate gene storage, amplification and expression.
  • the cloning vectors for use in the present invention include, but are not limited to, the following vectors: pBR322, pBR325, pACYC177, pUC8, pUC9, pUC18, pUC19> pLG339, pR290, pKC37, pKClOK SV 40, pBluescript II SK +/- , pQE, pGEM-T , pET series carrier.
  • the characteristics of the vector and the cloning method can be found in the "Molecular Cloning Practice Manual" prepared by the Cold Spring Harbor Laboratory, the contents of which are incorporated herein by reference.
  • Hosts for use in the present invention - vector systems include, but are not limited to, the following systems: bacteria - phage systems, bacteria - plasmid vector systems, yeast - yeast vector systems, mammals - viral systems (vaccinia virus, adenovirus, etc.) ), insect cells - rod ⁇ ! Canine virus system.
  • bacteria - phage systems bacteria - plasmid vector systems
  • yeast - yeast vector systems yeast - yeast vector systems
  • mammals - viral systems vaccinia virus, adenovirus, etc.
  • insect cells - rod ⁇ ! Canine virus system vaccinia virus, adenovirus, etc.
  • the method comprising: amplifying primer synthesis, ⁇ total RNA extraction, reverse transcription of mRNA, PCR amplification of the gene, recovery of the target gene fragment, cloning of the target gene fragment into the vector, and determination of the cDNA sequence of the scorpion fibrinolytic protein.
  • Amplification primer synthesis PCR oligonucleotide amplification primers were designed based on the published cDNA sequence of the scorpion fibrinolytic enzyme in GeneBank.
  • the upstream primer (P1) is 18 nucleotides long: ACATGGAACTTCCTCCCG
  • the downstream primer (P2) is 19 nucleotides long: ATC ACC AAC AACTAAACCG, and the primers are purified by PACE.
  • the first strand of cDNA was synthesized using Oligo(dT)20 as a primer according to Promega's Reverse Transcription Reaction kit. The reaction conditions were: 42 ° C for 1 hour; 95 ° C for 5 minutes; 3 ° C for 5 minutes.
  • the PCR reaction was carried out according to the PCR reaction system parameters given in the Reverse Transcription Reaction kit.
  • the first strand of cDNA was used as a template for PCR amplification.
  • the reaction parameters were: pre-denaturation at 95 °C for 4 minutes; denaturation at 94 °C for 1 minute, annealing at 55 °C for 1.5 minutes, extension at 72 °C for 2 minutes. Clock, 20 cycles; denaturation at 94 ° C for 1 minute, annealing at 55 ° C for 1.5 minutes, extension at 72 ° C for 2 minutes, 10 cycles; 72 ° C for another 10 minutes.
  • the target gene fragment was cloned into the vector: the recovered PCR product was ligated to the T vector, and the product was transformed into competent cell E. coli JM109, and the colony was quickly extracted and the plasmid was identified by restriction enzyme digestion. The positive clone obtained was obtained.
  • the subunit is Escherichia coli containing the gene of the present invention and is used for gene proliferation and preservation.
  • the gene mature peptide sequence is nucleotides 3 to 740, encoding 246 amino acids, and the remaining nucleotide sequences are non-coding regions.
  • the protein cDNA mature peptide stop codon is located at positions 741-743 of the gene, and the stop codon is TAA.
  • the invention provides the construction and expression of a Pichia pastoris genetically engineered strain expressing a plasmin gene, which can express a plasmin gene and obtain a genetically engineered plasmin
  • the protein is used for the preparation of a medicament for treating cardiovascular diseases and a health care product for preventing cardiovascular diseases by preventing thrombotic diseases.
  • the plasmin gene involved in the present invention can be expressed in a vector-host system including, but not limited to, a genetically engineered protein: an Escherichia coli system, a yeast expression system, a CHO serogroup expression system, a baculovirus expression system, Filamentous fungal expression system, Bacillus expression system.
  • the Pichia yeast expression system is a highly efficient eukaryotic expression system developed in recent years. Compared with other expression systems, it has the following advantages: (1) High expression, because Pichia pastoris grows fast, Moreover, the expression vector uses a strong promoter, the alcohol oxidase promoter, and the expression level of the foreign protein is high. (2) The stability is good.
  • the constructed engineering strain is very stable. (3) Easy to purify and easy to operate. The expression product is generally secreted extracellularly, and the protein of interest can be directly detected from the culture solution without renaturation, and the protein can be directly extracted from the supernatant. (4) High activity. The greatest benefit of exogenous protein expression in Pichia pastoris is the ability to perform post-translational processing of proteins, including disulfide bond formation, protein folding and glycosylation.
  • Pichia pastoris glycosylation of foreign proteins is closer to mammalian glycosylation, so the glycoprotein expressed by Pichia pastoris is considered to be more suitable for human body.
  • (5) Facilitate industrial production.
  • Pichia pastoris has ready-made, mature fermentation technology The expansion culture is carried out, and the Pichia pastoris expression system can grow using methanol as the sole carbon source, and the large-scale fermentation is convenient to produce, low in cost, and easy to industrialize.
  • the mound fibrinolytic enzyme cDNA of the present invention is derived from Lumbricus rubellus, which has a total of 741 bp and encodes 246 amino acids.
  • the present invention expresses the gene in a yeast system, produces a genetically engineered plasmin by fermentation, and detects the biological activity of the protein by a fibrin plate method.
  • a yeast system produces a genetically engineered plasmin by fermentation, and detects the biological activity of the protein by a fibrin plate method.
  • ⁇ fibrinolytic enzyme in E. coli, but its expression in yeast has not been found.
  • Pichia expression vectors include, but are not limited to, the following vectors: pPIC 6, pPIC 6 oc, pPIC Z, pPIC Z, pPIC9k, pPIC3.5k, pPA0815, pGAPZ, pGAP a Z; Pichia pastoris expressing strains include, but are not limited to, the following bacteria Lin: GS115, KM71, KM71H, X-33, SMD1168, SMD1168H.
  • upstream primer P1 upstream primer P1:
  • the PCR product was cloned into the T vector: the gene fragment in the PCR product was recovered and cloned into the pGEM-T vector, and the recombinant vector (pGEM-T-EFE2) was transferred into Escherichia coli JM109.
  • YPD 1% yeast extract, 2% peptone, 2% glucose
  • the primers are universal primers provided by Invitrogen expression vector, the sequence is upstream: GACTGGTTCCAATTGAC AAGC, downstream: GCAAATGGCATTC TGACATCC, the identified positive clone Pichia strain engineering strain is named Pichia pastoris X-33 ( pPIC6 a A-EFE ), the strain The deposit number of the China Type Culture Collection is CCTCC M204005.
  • Pichia pastoris there are two genes AOX1 and AOX2 encoding alcohol oxidase, which can be rapidly grown using sterol as the sole carbon source. When other carbon sources are present in the medium, the AOX gene is not expressed.
  • the foreign gene was cloned under the control of the AOX1 promoter, so it is necessary to use sterol as an inducer and the only carbon source to achieve high-level expression of the foreign protein. When inducing expression, it should be supplemented. Methanol is maintained at a level of 0.5%.
  • a method for inducing expression of a Pichia pastoris engineering strain includes, but is not limited to, the following mediums: MGY, MM, BMM, BMMY, The configuration of various media is a method that is very familiar to those skilled in the art, and details can be obtained by referring to relevant literature.
  • isolation and purification of proteins the principle of which is to separate from other proteins according to the physical and chemical properties of the protein of interest. For example, according to the molecular weight of the protein and the shape of the protein, centrifugation, ultrafiltration, gel filtration chromatography can be selected.
  • ammonium sulfate precipitation or acetone precipitation can be selected, depending on the isoelectric point.
  • Electro-focusing electrophoresis or isoelectric precipitation can be selected according to the hydrophobicity.
  • Hydrophobic interaction chromatography and reversed-phase HPLC can be used to select immobilized metal affinity chromatography based on the binding ability of protein to metal ions.
  • a method for expressing, isolating, and purifying a ⁇ plasmin gene in Pichia pastoris X-33 to obtain a genetically engineered protein is provided. The methods include: (1) induced expression of Pichia pastoris engineering strain; (2) isolation and purification of genetically engineered plasmin; (3) functional study and activity assay of genetically engineered plasmin protein.
  • Pichia pastoris engineering strains The expression method of Pichia pastoris engineering strains was as follows: Single colonies of recombinant bacteria were picked into 20 ml YPD liquid medium, cultured at 30 ° C, 250 rpm for activation; 10 ml after 16 hours The activated bacterial solution was transferred to 500 ml MGY liquid medium at 30 ° C, 250 rpm to increase the concentration; after 24 hours of concentration, the bacterial cells were centrifuged to collect the cells (1500 g, 10 minutes, 4 ° C); The body was resuspended in 1000 ml MM liquid medium for i-show expression, and sterol was added every 24 h to a final concentration of 0.5%; i-show 96 h, and the fermentation broth supernatant was collected by centrifugation (30000 g, 20 min, 4 ° C);
  • Methods for genetic engineering plasmin separation and purification include, but are not limited to, the following methods: Ni2+ column chromatography, ion exchange chromatography, affinity chromatography, condensation Glue filtration chromatography, electrophoresis recovery method, ultrafiltration method, ammonium acid precipitation method, and the like.
  • a method for isolating and purifying genetically engineered plasmin is provided, which is divided into three steps, the first is purification by Ni2+ column chromatography, and the genetically engineered plasmin protein has six consecutive histidine residues.
  • Ni2+ column chromatography is added to highly purified bovine enterokinase or recombinant endogut kinase for degradation reaction, and the non-natural amino acid residues in the fusion protein can be excised to obtain the genetic engineering protein identical to the amino acid composition of the natural protein. .
  • samples degraded by enterokinase were directly added to the QAE-Tyoypearl 550C column.
  • the genetically engineered plasmin was isolated and purified due to the different isoelectric points of different proteins. The purified product was verified by SDS-PAGE electrophoresis.
  • the bubble rise method of urokinase activity and the arginine lipase method (TAME) method for determining python antithrombotic enzyme but neither method is suitable for measuring ⁇ fibrinolytic enzyme, because plasmin has plasmin
  • the action of the original activator and proteolytic enzyme can exert both functions when the thrombus is dissolved, and the lytic activity of the enzyme is significantly greater than the kinase activity.
  • the fibrin plate method can objectively express the ability of plasmin to thrombolysis.
  • the fibrin plate contains plasminogen, which is converted to plasmin by the action of plasmin kinase.
  • fibrin occurs Hydrolysis, into a soluble small molecule peptide and amino acid, so that the plate at the loading site forms a translucent solution circle, and the activity unit of the enzyme is determined according to the size of the circle.
  • the size of the plate is related to the concentration range, so the activity of the fibrin plate can be regarded as the dual action of plasmin and activating enzyme.
  • the method has the advantages of simple operation, clear dissolution circle, easy observation, good repeatability and the like. Functional research method, bubble raising method, and arginine lipase method of genetically engineered plasmin protein applied to the gene of the present invention.
  • a method for determining the biological activity of genetically engineered plasmin by a fibrin plate method is provided.
  • the activity unit of the genetically engineered strain fermentation broth of the present invention exceeds 3,390,000 U/L by activity assay.
  • Further provided in one embodiment of the invention is the construction of a genetically engineered strain, the strain being Pichia pastoris X-33 (pPIC 6A-EFE), CCTCC M204004.
  • a method for expressing a sputum plasmin gene in Pichia pastoris by expressing a sputum plasmin gene in Pichia pastoris X-33 using a pPIC 6A vector including: (1) ⁇ fibrinolytic enzyme PCR amplification of the gene; (2) cloning of the PCR product into the T vector; (3) construction of the shuttle expression plasmid; (4) construction of the yeast genetically engineered strain expressing the plasmin gene, the steps of which are as follows: (1) PCR amplification of ⁇ fibrinolytic enzyme gene: Primer was designed based on the EFE mature peptide and the cloning site on the expression vector pPIC6A using the T vector plasmid containing the plasmin gene as a template, and the upstream primer P1: GAATTCGCATGGAACTTCCTCCCG (underlined) The endonuclease EcoR I site), the downstream primer P2: TCTAGACGGTTTAGTTGTTGGTGAT
  • the PCR product was cloned into the T vector: the gene fragment in the PCR product was recovered and cloned into the pGEM-T vector, and the recombinant vector (pGEM-T-EFE2) was transferred into Escherichia coli JM109.
  • the primers are universal primers provided by Invitrogen expression vector, the sequence is upstream: GACTGGTTCCAATTGACAAGC, downstream: GCAAATGGCATTC TGACATCC, the positive clone identified is named Pichia pastoris X-33 (pPIC 6A-EFE), the preservation number of the China Center for Type Culture Collection For CCTCC M204004, there are two genes encoding AOT1 and AOX2 encoding ethanol oxidase in Pichia pastoris, which can be rapidly grown using methanol as the sole carbon source. When other carbon sources are present in the medium, the AOX gene is not expressed.
  • the foreign gene was cloned under the control of the AOX1 promoter, so it is necessary to use sterol as an inducer and the only carbon source to achieve high-level expression of the foreign protein.
  • Methanol is maintained at a level of 0.5%.
  • a method for inducing expression of Pichia pastoris engineering strain is provided, and the medium for inducing expression of Pichia pastoris engineering strain includes However, it is not limited to the following media: MGY, MM, BMM, BMMY, and the configuration method of various media is a method which is very familiar to those skilled in the art, and details can be obtained by referring to relevant literature.
  • Pichia pastoris engineering strain was as follows: Single colonies of recombinant bacteria were picked into 20 ml YPD liquid medium and cultured at 30 °C and 250 rpm for activation; after 16 hours, 10 ml of activated bacteria solution was transferred to 500 ml MGY liquid medium. The cells were concentrated at 30 ° C and 250 rpm; the cells were concentrated for 24 hours to collect the cells by centrifugation (1500 g, 10 min, 4 ° C); the collected bacteria were suspended in 1000 ml of MM liquid medium for induction expression.
  • Methanol was added every 24 hours to a final concentration of 0.5%; after 96 hours of induction, the cells were collected by centrifugation (30000 g, 20 minutes, 4 °C), and the cell extracts were prepared by glass bead disruption for activity determination.
  • the current measurement method can only refer to the activity determination method of other thrombolytic drugs.
  • the bubble rise method of urokinase activity and the arginine lipase method (TAME) method for determining python antithrombotic enzyme but neither method is suitable for measuring ⁇ fibrinolytic enzyme, because plasmin has plasmin
  • the action of the original activator and proteolytic enzyme can exert both functions when the thrombus is dissolved, and the lytic activity of the enzyme is significantly greater than the kinase activity.
  • the fibrin plate method can objectively express the ability of plasmin to thrombolysis.
  • the fibrin plate contains plasminogen, which is converted to plasmin by the action of plasmin kinase.
  • fibrin occurs Hydrolysis, into a soluble small molecule peptide and amino acid, so that the plate is formed into a translucent solution circle, and the enzyme activity unit is determined according to the size of the circle.
  • the size of the plate is related to the concentration range, so the activity of the fibrin plate can be regarded as the dual action of plasmin and activating enzyme.
  • the genetic engineering method can obtain a large amount of fibrinolytic enzyme by simple fermentation, and separate and purify the process cartridge, and the product has high purity and low cost. The method has the advantages of simple operation, clear dissolution circle, easy observation, good repeatability and the like.
  • a method for determining the biological activity of plasmin by a fibrin plate method wherein the activity unit of the genetically engineered strain fermentation broth of the present invention exceeds 2,900,000 U/L.
  • a genetically engineered recombinant Pichia pastoris X-33-pPIC6o;-EFE is provided.
  • the invention further provides the use of the genetically engineered plasmin protein for the preparation of a medicament for treating cardiovascular and cerebrovascular diseases.
  • the genetic engineering protein involved in the invention has wide application in the research and development of medical, health and health care drugs, and can be used for preparing lyophilizates, water injections, tablets, suppositories, sprays, etc.
  • the genetic engineering drugs involved in the invention have the following advantages: (1) The oral administration of sputum fibrinolytic enzyme preparation is slow, cannot be intravenously, and cannot be used for treating acute Cardiac and cerebral embolism diseases; (2) The production process of capsules is complicated, and it is necessary to artificially culture large amounts of cockroaches. The process of separating and purifying plasmin from worms is cumbersome, and the products may contain various small molecular peptides or other chemicals. Causes allergic reactions in the body.
  • the genetic engineering method can obtain a large amount of fibrinolytic enzyme by simple fermentation, and separate and purify the process cartridge, and the product has high purity and low cost.
  • the genetic engineering drugs of the present invention have the following advantages:
  • Streptokinase SK and urokinase UK are first-generation thrombolytic drugs that directly or indirectly activate plasminogen to convert it into fibrinolytic fibers. Lysozyme.
  • SK is an exogenous plasminogen activator extracted from ⁇ -type hemolytic streptococcus culture medium and is an indirect plasminogen activator.
  • UK is a serine protease isolated from human urine and can also be obtained from human embryonic kidney cell culture fluid. There are two unavoidable problems in the actual use of these two enzymes: First, their specificity is poor, which often leads to systemic activation of plasminogen in the dissolution of thrombus, resulting in indiscriminate digestion of coagulation proteins.
  • SK is a non-enzymatic protein and the biggest problem in its use is its antigenicity. Almost all people contain anti-SK antibodies more or less in their blood. The presence of antibodies on the one hand "neutralizes" the drug, which reduces the efficacy, and on the other hand may cause an allergic reaction.
  • tissue type plasminogen activator tPA is a serine protease mainly produced by vascular endothelial cells, which catalyzes the inactive plasminogen to become active fibrinolysis. Enzyme to dissolve the thrombus.
  • Normal human tPA has a plasma concentration of about 5 ng/ml and a half-life of 5 minutes.
  • the specificity of tPA for plasminogen present in the thrombus fraction is considered as a second-generation thrombolytic drug.
  • tPA produced by genetic engineering has begun to be used in clinical practice, showing a certain superiority. Sex.
  • Sex due to the short half-life, it is necessary to repeatedly use a large amount of drugs to work, thereby increasing the possibility of systemic fibrinolysis leading to internal bleeding, and its high cost limits its widespread use.
  • thrombolytics In addition to UK, SK and tPA, snake venom antithrombin, single-chain urokinase-type plasminogen activator (pro-UK), acetyl-fibrinolytic Enzymatic streptokinase activating complex (APSAC) and the like.
  • pro-UK single-chain urokinase-type plasminogen activator
  • APSAC acetyl-fibrinolytic Enzymatic streptokinase activating complex
  • Bio-extracted thrombolytic drugs require activated carbon adsorption during the production process, mainly for decolorization, adsorption of heat sources, removal of shields and filtration.
  • the process protein has high purity, pure quality, no pyrogen, avoids decarbonization process and activated carbon pollution to the environment, and has low production cost, and can be directly used for producing water for injection and powder.
  • the genetically engineered plasmin produced by the present invention may be mixed with a pharmaceutically acceptable carrier (such as an excipient, a filler, an absorption enhancer, etc.) or mixed with other drugs, and may be prepared according to a conventional method in the pharmaceutical field.
  • the desired dosage form can be administered by oral administration, injection or the like. These dosage forms include, but are not limited to, lyophilizates, aqueous injections, tablets, suppositories, sprays.
  • the genetic engineering drugs involved in the invention can be applied to the treatment or adjuvant treatment of cardiovascular and cerebrovascular diseases, including but not limited to: ischemic encephalopathy, acute heart-month infarction combined with hyperfibrinogenemia, coronary heart disease, Hyperviscosity caused by coronary heart disease, coronary heart disease angina pectoris, unstable angina pectoris, fibrinogen and platelet aggregation hyperplasia, the genetic engineering drugs involved in the present invention can also be applied to the treatment of some diseases related to hemorheology And adjuvant therapy, including but not limited to Meniere's disease, sudden deafness, pulmonary heart disease, diabetes, retinal vein occlusion, nephrotic syndrome.
  • cardiovascular and cerebrovascular diseases including but not limited to: ischemic encephalopathy, acute heart-month infarction combined with hyperfibrinogenemia, coronary heart disease, Hyperviscosity caused by coronary heart disease, coronary heart disease angina pectoris, unstable angina pectoris, fibrinogen and
  • the genetically engineered protein according to the present invention can be used as a main active ingredient for the production of health care products, for preventing thrombotic diseases, and for improving cardiovascular and cerebrovascular health.
  • the above-mentioned medicine or other biological products with genetically engineered plasmin as the main active ingredient, the production process or method thereof is familiar to those skilled in the art, and the production process should be In accordance with relevant national laws and regulations, including the Pharmacopoeia of the People's Republic of China
  • a method for preparing a genetically engineered plasmin thrombolytic drug preparation is provided, and a thrombolysis and a protective effect against cerebral ischemia are performed in an animal model, and the results indicate that the present invention relates to
  • the genetically engineered plasmin has a good thrombolytic effect and has a protective effect on the cerebral ischemia of the test animal.
  • the present invention provides a genetically engineered strain, Pichia pastoris X-33 (pPIC6 a A-EFE), deposited by: China Center for Type Culture Collection, Date of Deposit: January 12, 2004, Deposit No.: CCTCC No : M204005.
  • the present invention provides a genetically engineered strain, Pichia pastoris X-33 (pPIC6 A-EFE), deposited by: China Center for Type Culture Collection, Preservation Period: January 12, 2004, Deposit No.: CCTCC No : M204004.
  • the invention is further illustrated by the following examples in conjunction with the accompanying drawings and embodiments, which are not intended to limit the scope of the invention.
  • the media and molecular biology methods of operation in all of the examples are familiar to those skilled in the art and can be found in "Molecular Cloning” by Sambrook et al. (Handbook of Reality, Cold Spring Harbor, 1989) and "Guidelines for Editing Molecular Biology".
  • the PCR oligonucleotide amplification primers were designed according to the published sequence of the plasminogen plasmin cDNA in GeneBank.
  • the upstream primer is 18 nucleotides long, ACATGGAACTTCCTCCCG, and the downstream primer is 19 nucleotides long, ATCACCAACAACTAAACCG.
  • the primers were synthesized by Shanghai Shenggong Bioengineering Co., Ltd. and purified by PAGE.
  • the first strand of reverse transcribed cDNA was synthesized according to Promega's Reverse Transcription Reaction kit, and the first strand of cDNA was synthesized using Oligo(dT)20 as a primer.
  • the specific steps are as follows: Add the following reagents to a DEPC-soaked and sterilized PCR reaction tube: 25 mM MgCl 2 12 ⁇ 1; buffer 6 ⁇ 1; 10 mM dNTP 6 ⁇ 1; Recombinant RNasin Ribonuclease Inhibitor 2 ⁇ 1; AVM reverse transcriptase 4 ⁇ 1; Oligo (dT) 20 6 ⁇ 1; ⁇ total RNA 15 ⁇ ⁇ ; sterilized double distilled water 9 ⁇ ⁇ Reaction conditions: 42 ° C 1 hour; 95 ° C 5 Minutes; 3 °C for 5 minutes.
  • the reaction components are: buffer 2.5 ⁇ 1; 25 mM MgCl 2 1.5 ⁇ l; l OmM dNTP 2 ⁇ 1; upstream primer ⁇ ⁇ ; downstream primer ⁇ ; mRNA reverse transcription product 15 ⁇ 1; RT-PCR enzyme mixture 2 ⁇ 1; sterilized double distilled water 5 ⁇ 1 .
  • the reaction parameters are: buffer 2.5 ⁇ 1; 25 mM MgCl 2 1.5 ⁇ l; l OmM dNTP 2 ⁇ 1; upstream primer ⁇ ⁇ ; downstream primer ⁇ ; mRNA reverse transcription product 15 ⁇ 1; RT-PCR enzyme mixture 2 ⁇ 1; sterilized double distilled water 5 ⁇ 1 .
  • the reaction parameters are: buffer 2.5 ⁇ 1; 25 mM MgCl 2 1.5 ⁇ l; l OmM dNTP 2 ⁇ 1; upstream primer ⁇ ⁇ ; downstream primer ⁇ ; mRNA reverse transcription product 15 ⁇ 1; RT-PCR enzyme mixture 2 ⁇ 1; sterilized double distilled water 5
  • the target gene fragment was recovered according to the instructions of the PCR product recovery kit of Shanghai Huasheng Bioengineering Co., Ltd., as follows: Take 30 1 PCR reaction product and add 1.4 ml of PB solution (provided by the kit:), and the mixture was transferred to the adsorption column.
  • Centrifuge for 15 seconds, discard the waste liquid add 400 ⁇ l of PB solution to the adsorption column, let stand for 1 minute, centrifuge for 15 seconds, discard the waste liquid; add 500 ⁇ 1 ⁇ 1 solution (provided by the kit), centrifuge for 15 seconds, discard The waste liquid was repeated once; 300 ⁇ l of ⁇ 1 solution (provided by the kit) was added to the adsorption column, and after standing for 1 minute, it was centrifuged for 30 seconds, and the centrifugation liquid was collected and stored at -20 °C.
  • the target gene fragment was cloned into the vector, and the gene fragment was cloned into pUCm-T vector, which was purchased from Shanghai Shenggong Bioengineering Co., Ltd.
  • the reaction components were as follows: PCR product 5 ⁇ l; pUCm-T vector 1 ⁇ l; 10XT4 DNA ligase buffer ⁇ ; T4 DNA ligase 1 ⁇ l; deionized water 2 ⁇ l; total volume 10 ⁇ l, overnight at 4 °C.
  • Preparation of competent cells A single JM109 colony was picked, inoculated into 3 ml of ampicillin-free LB medium, and cultured at 37 ° C overnight, and the above-mentioned bacterial solution was inoculated in a ratio of 1:100 and then inoculated into 50 ml of LB medium. After shaking at 37 ° C for 3 hours, when the OD value of the bacterial solution was 0.6, centrifuge at 5,000 rpm for 8 minutes at 4 ° C, discard the supernatant, suspend the pellet with 0.1MCaCl 2 , centrifuge at 5000 rpm for 8 minutes, discard the supernatant, and precipitate with an appropriate amount of 0.1 M.
  • the CaCl 2 was resuspended and placed in a water bath for 6 hours. Transformation of the ligation product: Take the above-mentioned ligation product 5 ⁇ 1 into a 100 ⁇ ⁇ competent cell water bath for 60 minutes, heat shock at 42 ° C for 90 seconds, then place in an ice bath for 2 minutes, add ampicillin-free LB medium 0.9 ml Incubate at 37 ° C for 1 hour, add 200 ⁇ l of bacterial solution to 40 ⁇ l 20 mg/ml 5-bromo-4chloro-3-indole (D-galactoside (X-gal)) and 4 ⁇ l 0.1 ⁇
  • the propyl thiogalactoside (IPTG) was mixed with ampicillin LB, and cultured at 37 ° C for 16 hours.
  • the bacterial solution was added to a 1.5 ml centrifuge tube, centrifuged at 5000 rpm for 8 minutes, the supernatant was discarded, and the precipitate was suspended in a 15 ⁇ M suspension (50 mM glucose, 25 mM Tris-HCl, 10 mM EDTA 5 PH8.0), and a 200 ⁇ l lysate ( 0.2M NaOH, 1% SDS), after 5 minutes in a water bath, add 150 ⁇ M ⁇ neutralizing solution (3.0 MKAc, pH 4.8), ice bath for 10 minutes, centrifuge at 12000 rpm for 10 minutes.
  • a 15 ⁇ M suspension 50 mM glucose, 25 mM Tris-HCl, 10 mM EDTA 5 PH8.0
  • a 200 ⁇ l lysate 0.2M NaOH, 1% SDS
  • the gene mature peptide sequence is ranked 3-740 nucleotides, and the remaining nucleotide sequence is a non-coding region.
  • the protein cDNA mature peptide stop codon is located at positions 741-743 of the gene and the stop code is TAA.
  • the isoelectric point (IP) of the protein predicted by the lysozyme cDNA sequence was 4.15, and the protein was acid 'I'.
  • the gene fragment of the present invention may be modified, such as a certain nucleotide sequence of the mutated gene, but does not affect the amino acid sequence of the encoded protein;
  • the amino acid or nucleotide sequence involved in the invention these modifications will not Affects the biochemical properties of proteins, advanced structures, and biological functions of proteins, such as: secretion of signal peptides at the N-terminus or C-terminus of a protein or peptide, plus the addition of appropriate linkers (eg, 6 histidines) ) Facilitate protein extraction and purification.
  • nucleotide sequence may be artificially synthesized, and the synthesized nucleotide sequence may differ from the sequence provided by the present invention, but the encoded protein is identical to the protein sequence involved in the present invention, and these possible synthetic sequences are as follows: atggarytnccnccnggnacnaarathgtnggnggnathgargcnmgnccntaygarttycc ntggc argtnwsngtnmgnmgnaarwsnwsngaywsnc ayttytgyggnggnwsnath athaaygaymgntgggtngtntgygcngcncaytgyatgcarggngargcnccngcnytn gtnwsnytngtngtnggngarcaygaymgnwsngcnwsnacngtnmgncaracnc ayg
  • ATGGAACTTCCTCCCGGAACA (underlined is the endonuclease EcoR I site).
  • Downstream primer P2 GTCTAGATTAGTTGTTGGTGATGATGTC GGTGATCCATGCAGCAT (underlined is the endonuclease Xba I site:).
  • the primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd., and the synthesized products were purified by PAGE.
  • the reaction components were: buffer 2.5 ⁇ ⁇ ; 25 mM MgC12 1.5 ⁇ ⁇ ; 10 mM dNT P 2 ⁇ 1; upstream primer ⁇ ⁇ ⁇ ; downstream primer ⁇ ⁇ ⁇ ; pUCm-T-EFE Shield granule 1 ⁇ 1; LA Taq DNA polymerase 2 ⁇ 1; sterilized double distilled water 19 ⁇ l.
  • the reaction parameters were: pre-denaturation at 9 5 °C for 4 minutes; denaturation at 94 °C for 1 minute, annealing at 55 °C for 1.5 minutes, extension at 72 °C for 2 minutes, 30 cycles; and extension at 72 °C for 10 minutes.
  • Pichia expression vectors for selection on blasticidin and purification of recombinant proteins manual Pick Pichia pastoris X-33 single colony inoculated into 5 ml of YPD medium, incubate at 30 ° C for 250 rpm shaker overnight, inoculate 200 ml of YPD liquid medium in 1% inoculum, and incubate for 30 to 5 hours at 30 ° C 250 rpm shaker.
  • the concentration of the cells reached 0.8-1.0, centrifuged at 1500g for 10 minutes, the pellet was resuspended in 10 ml of sterilized double distilled water, centrifuged at 1500 g for 10 minutes, and the pellet was resuspended in 0.4 ml of 100 mM LiCl and transferred to a sterilized 1.5 ml centrifuge tube. Centrifuge at 12000g for 15 seconds, discard the supernatant, resuspend in 0.16 ml of 0.1 M LiCl, and set aside.
  • Recombinant scutellum pPIC6 o A-EFE was extracted and cut linearly with Sac I, and the empty plasmid pPIC6 c A was also digested as a control. Take 50 ⁇ l of the above competent cells, centrifuge at 12000g for 15 seconds, carefully pipet the supernatant with a pipette, and add the following components in order: 240 ⁇ 1 50% PEG; 36 ⁇ 1 1 M LiCl; 25 l 2 mg/ Ml salmon sperm DNA; 50 ⁇ l (5-10 g) The linearized recombinant plasmid pPIC6aA-EFE 0 was linearized with the empty plasmid PIC6 ⁇ ⁇ as a control.
  • Yeast genomic DNA preparation method Take 1.5 ml of culture solution at 4 000 r / min for 10 minutes from the heart. The cells were ground and broken with liquid nitrogen. Add 7 mL of DNA buffer (100 mmol/L Tris HC1, pH 8. 0, 10 mmol/L EDTA, 1% SDS) and mix at 65 °C for 1 h. Centrifuge for 15 minutes at lO OOO r/min. The supernatant was extracted twice with phenol and centrifuged at 5 000 r/min for 7 minutes each time. It was extracted once with chloroform and centrifuged at 5 000 r/min for 7 minutes. The mixture was precipitated with 2.5 times ethanol (0.3 mol/L sodium acetate, ⁇ 5. 2), and centrifuged at 10 000 r/min for 10 minutes. Wash with 70% ethanol, blow dry with hair dryer, add TE or water suspension.
  • DNA buffer 100 mmol/L Tris HC1, pH 8. 0, 10 mmol/L EDTA, 1% SDS
  • PCR identification method Primers are universal primers provided by Invitrogen expression vector, the sequence is upstream: G ACTGGTTCC AATTG AC AAGC; downstream: GCA AATGGCATTC TGACATCC.
  • the PCR was carried out in the usual manner, and the amplification conditions were: 94 ° C for 45 seconds, 54 ° C for 1 minute, 72 ° C for 1 minute, and 40 cycles.
  • the amplified product was checked for correctness by using 0.8% agarose, and the results are shown in Fig. 3.
  • the positive clone obtained was named Pichia pastoris X-33 (pPIC6aA-EFE), and the strain was deposited at the China Center for Type Culture Collection: CCTCC M204005.
  • Example 3 Expression, isolation, purification of genetically engineered plasmin protein
  • Fermentation process of Pichia pastoris engineering strain X-33 (pPIC6aA-EFE): a. L single colony of recombinant bacteria into 20ml YPD liquid medium (1% yeast extract, 2% peptone, 2% glucose) , 30 ° C, 250 rpm culture for activation; b. After 16 hours, take 10ml of activated bacterial solution to transfer 500ml MGY liquid medium liquid medium 30 ° C, 250 rpm culture enrichment; c. After enrichment for 24 hours The bacterial cells were collected by centrifugation (1500 g, 10 min, 4); d.
  • YPD liquid medium 1% yeast extract, 2% peptone, 2% glucose
  • Double distilled water to wash the outer surface of the dialysis bag, immersing the dialysis bag containing the concentrated fermentation broth in the same volume as the original fermentation broth Lx BINDING BUFFER (5mM imidazole+0.5M NaCl+20mM Tris ⁇ HCl PH 7.9) overnight to balance the pH and ion concentration of the fermentation broth; c with step a, concentrate to a volume of about 1/10 of the original fermentation broth; d Add lx BINDING BUFFER to the dialysis bag and dilute the concentrated sample by 4 times; e. Same as step c; f.
  • Ni2+ column chromatography a. Ni2+ column Wash the column bed with 10 beds of 1 x BINDING BUFFER, 10 beds of sterile water, supplement the Ni2+ with 10 beds of lx CHARGE BUFFER (5Mm NiS04), and then clean the column bed with 10 beds of l xBINDING BUFFER to prepare for the start of adsorption chromatography. (1 bed is the volume of the medium in the column); b. The concentrated sample is passed through the Ni2+ column in a cyclic manner by a peristaltic pump, so that the sample with 6 x HIS is fully combined with Ni2+, and after being collected from the Ni2+ column outlet, it is reacted with Ni2+ after overnight.
  • a sample through a leak
  • the number of eluted beds and the concentration of the elution solution vary flexibly depending on the total protein content and the binding ability of the non-target protein to Ni2+. Note: If the above formula is used, the eluted heteroprotein solution is 60 mM sodium saliva. e.
  • the target protein was eluted with 1 x ELUTE BUFFER, and collected in steps.
  • the volume of each bed (actually less than the volume of the bed) was one tube, and the distribution of the target protein in the eluate was determined by SDS-PAGE.
  • Purification process (operating at a temperature below 4 °C): Add the dialyzed sample to the column and collect the effluent; wash the column with 3 X bed volume (150 ml) of buffer A; buffer A Flush B (25 mM Hepes/pH 8.0, 300 mM aCl, 5 mM EDTA) was eluted through a preparative NaCl concentration gradient buffer through a linear gradient former. (Buffer A 200 liters; Buffer B 200 ml); Eluted samples were collected in steps of 1 ml volume per part using an automated sample collector. The protein elution peak was monitored by OD280 nm ultraviolet light. After confirming the main protein peak sample tube containing plasmin by SDS-PAGE, these samples were mixed.
  • Electrophoresis results as shown in Figure 4.
  • Example 4. Functional study and activity assay of genetically engineered plasmin protein
  • the biological activity of plasmin was determined by fibrin plate method.
  • the activity unit of the fermentation broth exceeded 3 E+06 U/L by activity measurement.
  • Figure 5 shows the fibrinolysis circle during the measurement.
  • the detection method can refer to the method of Hao Suli et al. [Hao Suli, Shen Jia, Research on the Bioactivity Detection Method of Prion Kinase, Chinese Pharmacy, Vol. 10, No. 6, 1996].
  • the specific operation method is as follows: . .
  • primers were designed based on the cloning site of EFE mature peptide and expression vector pPIC6A, upstream primer PI: GAATTC GCA TGG AAC TTC CTC CCG ( Underlined is the endonuclease EcoR I site), downstream primer P2: TCT AGA CGG TTT AGT TGT TGG TGA T (underlined is the endonuclease Xba I site).
  • the primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd., and the synthesized products were purified by PAGE.
  • the reaction components were: buffer 2.5 ⁇ 1; 25 mM MgCl 2 1.5 ⁇ 1; 10 mM dNTP 2 ⁇ 1; upstream primer 1 ⁇ 1; downstream primer 1 ⁇ 1; pUCm-T-EFE plasmid 1 ⁇ 1; LA Taq DNA polymerization Enzyme 2 ⁇ 1; Sterilized double distilled water 19 ⁇ 1 .
  • the reaction parameters were: pre-denaturation at 95 °C for 4 min; denaturation at 94 °C for 1 min, annealing at 55 °C for 1.5 min, extension at 72 °C for 2 min, 30 cycles; and extension at 72 °C for 10 min.
  • the dephosphorylated linearized pPIC6A empty vector and the double-digested EFE were subjected to T4 DNA ligase overnight at 4 ° C to obtain a recombinant expression vector PIC6A-EFE, ⁇ !
  • the recombinant vector was transferred to E. coli JM109 for amplification.
  • the white colonies were picked to quickly extract the plasmid for enzyme digestion, and the results of the digestion of the digested product by 1% agarose are shown in Fig. 3.
  • the concentration of the cells reached 0.8-1.0, centrifuged at 1500g for 10 minutes, the pellet was resuspended in 10 ml of sterilized double distilled water, centrifuged at 1500 g for 10 minutes, and the pellet was resuspended in 0.4 ml of 100 mM LiCl and transferred to a sterilized 1.5 ml centrifuge tube. Centrifuge at 12000g for 15 seconds, discard the supernatant, resuspend in 0.16 ml of 0.1 M LiCl, and set aside. The plasmid pPIC6A-EFE was recombined, and was linearly cut with Sacl. The same was used to cut the broad-spectrum pPIC6A as a control.
  • the linearized recombinant plasmid pPIC6A-EFE and the empty plasmid pPIC6A were transformed into X-33 yeast strain by biochemical methods (refer to Picliia expression vectors for selection on blasticidin and purification of recombinant proteins manual, Invitrogen). Incubate on YPD solid medium containing Blasticidin s Hcl (300 mg/ml) for 2 to 3 days until a single colony appears. (5) The positive transformants were screened and 10-20 single colonies were cultured in YPD liquid medium at 30 ° C, shaken at 250 rpm until late logarithmic growth, and the genome was extracted for PCR identification of recombinants.
  • the obtained male '1 ⁇ sheng recombinants are both Mut+.
  • Yeast genomic DNA preparation method 1.5 ml of culture solution was centrifuged at 4 000 r/min for 10 min. The bacteria are hard to break with liquid nitrogen. Add 7 ml DNA buffer (100 m mol/L Tris-HCl, pH 8. 0, 10 m mol/L EDTA, 1% SDS). Mix well, keep 65 V for 1 h. Centrifuge at 10 000 r/min for 15 min. The supernatant was extracted twice with phenol and centrifuged at 5 000 r/min for 7 min each time. It was extracted once with chloroform and centrifuged at 5 000 r/min for 7 min.
  • the mixture was precipitated with 2.5 times ethanol (0.3 mol/L sodium acetate, ⁇ 5. 2), and centrifuged at 10 000 r/min for 10 min. Wash with 70% ethanol and blow dry with a hair dryer. Add TE or water suspension, force RNase.
  • PCR identification method Primers are universal primers provided by Invitrogen expression vector, the sequence is upstream: GACTGG TTCCAATTGACAAGC; downstream: GCA AATGGCATTC TGACATCC.
  • the PCR was carried out according to the conventional method.
  • the amplification conditions were: 94 "C 45 s , 54 ° C for 1 min , 72 ° C for 1 min , 40 cycles .
  • the product was widely detected with 0.8% agarose.
  • Example 6 Induced expression of genetically engineered plasmin protein
  • a single colony of the recombinant bacteria is picked up into 20 ml of YPD liquid medium (1% yeast extract, 2% peptone, 2% glucose), and cultured at 30 ° C, 250 rpm for activation;
  • the volume of the compacted packet is measured before the yeast digestion buffer is resuspended, and all the following steps are performed at 4 ° C; (7) 1 volume of glass bead sterilizing buffer (20mmol/L Tris-Cl, pH 7.9, 10 m mol/L MgCl 2 , 1 m mol/L EDTA, 5% glycerol, 1 m mol/L DTT, 0.3 m mol/L acid, 1 m mol/L PMSF)
  • Example 7 Activity assay of genetically engineered plasmin protein The biological activity of genetically engineered plasmin was determined by fibrin plate method. The activity unit of the fermentation broth exceeded 2.9E+06 U/L by activity assay. Figure 6 shows the measurement For the detection of the melting circle, the method of detection can be referred to the method of Hao Suli et al.
  • the medium was adjusted to pH 6.0 with 30% ammonia water, and sterilized at 121 °C for 20 min. After sterilization, PTMi was added to the filter to a final concentration of 4.35/L.
  • PTM 2 salt solution PTM 2 salt solution:
  • the solution was sterilized by filtration through a 0.22 ⁇ : ⁇ filter.
  • Inoculation amount 10% MGY seed fermentation stage control: Fermentation tank induced Pichia pastoris expression of genetic engineering protein EFE process is divided into two stages: the expansion of engineering bacteria and the induction of expression of the target protein.
  • a single colony was picked from freshly streaked plates at 30 ° C in 500 MGY medium, and cultured on a 300 rpm shaker for about 36 h to an OD 6 QO of 15 as a seed fungus.
  • the seed fungus was inoculated into 5 liters of the sterilized basic salt medium for concentration enhancement.
  • the temperature is controlled at 29 °C, and the ventilation and rotation speed are adjusted so that the dissolved oxygen is not less than 25%.
  • the rapid rise of dissolved oxygen indicated that the glycerol in the medium had been exhausted.
  • Glycerol (50% w/v, 12 mL/LPTMl) was initially supplied at a rate of 75 mL/h, which lasted for approximately 6 h to a wet bacterial weight of 200 g/L.
  • the % principle adjusts the methanol flow rate.
  • the initial acceleration of methanol (100%, 12mL/L PTMl) is 15mL / h for the first 4 hours, 40 mL / h for the next two hours, and the final acceleration is controlled at 50mL / h.
  • the speed was maintained until the induction of expression was about 83.5 h, and the fermentation was completed. At this time, the wet bacteria weight was 270 g/L.
  • the fermentation broth was collected, centrifuged at 4000 rpm for 15 min, and the supernatant was collected and stored at -20 °C until use.
  • the wet bacterial weight and the activity of the expressed product per unit volume were sampled every 6 hours.
  • the wet bacterial weight reached 230 g/L after 48 hours of induction, and the activity of the expressed product was detected, indicating that the engineered protein had begun to express.
  • the fermentation broth was ultrafiltered and had fibrinolytic activity after salting out (NH4) 2 S0 4 .
  • NH4 salting out
  • the fibrinolytic activity per liter of the 16-hour fibrin plate contained about 1.5 million units per liter.
  • Example 9 Gene engineering plasmin preparation of thrombolytic drugs and
  • Preparation of oral thrombolytic drugs will be purified plasmin (method see Example 2, Example 5, Example 8) in buffer (23 g glycine / liter, 1.6 g disodium hydrogen phosphate / liter, 0.55 g phosphoric acid Sodium dihydrogen/liter, pH 7.0) After dialysis, it was concentrated by ultrafiltration to a concentration of 1 gram per ml, and sterilized by filtration through a membrane having a pore size of 0.22 ⁇ m. Then, about 1 liter of sample solution (about 300000 vitality unit) bottling, freeze-drying, finished product. When used, the sample is preferably dissolved in 1 liter of medical water for bioassay and injection.
  • the genetic engineering plasmin sample size is: 300000 U genetically engineered plasmin; 23 mg glycine; 1.6 mg disodium hydrogen phosphate; 0.55 mg sodium dihydrogen phosphate. Also equipped with 5 ml medical grade water. The genetically engineered fermentation product was disrupted (see Example 5 for the method), and then the supernatant was collected in buffer (23 g glycine/liter, 1.6 g disodium hydrogen phosphate/liter, 0.55 g sodium dihydrogen phosphate/liter, pH 7. 0) Dialysis, finally concentrated to a concentration of 1 mg per ml by ultrafiltration, and sterilized by filtration through a 0.22 ⁇ m pore size membrane.
  • the filtrate can be directly administered to the patient as the most oral liquid, or about 1 ml of the filtrate (about 300,000 viability units), lyophilized, and finished into a powder. When used, the water is dissolved in warm water.
  • Example 10 Application of genetically engineered plasmin protein preparation The experimental method of rat anticoagulation was carried out with reference to the literature of Chen et al. [Chen Health, Wang Lei, Li Wei, Guo Feng, Yang Jun, Apricot Flavonoids and Genetically Engineered Plasmin Anticoagulant thrombolytic effect, Heart Journal 2001, 13(4): 308-309], the entire disclosure of which is incorporated herein by reference. Thirty male Kunming mice were randomly divided into 3 groups and weighed 20 soil and 2 g.
  • each group was injected with 5000 U/kg genetically engineered plasmin, 0.2 mg of aspirin, and the control group was injected with 0.5 mL of physiological saline. 2 hours after the third day of medication Experiment. The tail of the mouse was cut 1 cm, and the serum was removed by filter paper at intervals of 10 s. The blood was not squeezed and the bleeding time of the mice was recorded. The experimental results (see Table 1) showed that the bleeding time of the genetically engineered plasmin group and the aspirin group was prolonged, and the genetically engineered plasmin group and the control group were significantly more significant, indicating that the genetically engineered plasmin has good resistance. Coagulation. Experimental mouse tail bleeding time comparison table
  • the rats were anesthetized with 10% chloral hydrate (300 mg/kg), fixed in the supine position, and the midline incision was made in the neck. The right common neck was exposed. From the bifurcation to the head end, the occipital artery and the thyroid gland were ligated and cut. The superior artery was ligated and cut at the distal end of the external carotid artery, and was freed for use. Separate the internal carotid artery, use a silk thread to make a loose buckle at the base of the external carotid artery, clip the neck to move the moon and the neck and move the moon forever. Use the thin wire to move through the neck outside the main trunk incision, and slow down.
  • the sham operation group exposed the left middle cerebral artery and the left common carotid artery without ligation.
  • Each of the administration groups was intravenously injected with genetically engineered plasmin 10 minutes before ligation, and the model group was given 0.5 ml of physiological saline.
  • a p ⁇ 0.05
  • b ⁇ ⁇ 0.01
  • c p ⁇ 0.001
  • d p ⁇ 0.05
  • Level 5 Level I is no neurological work Can damage, score 1 point; Grade II only mild dysfunction, manifested as walking without support, but the gait is not stable, the tail injury of the contralateral limbs during tailing, 2 points; Level III has moderate function Obstruction, manifested as standing under the support, not persistent, flexion of the contralateral forelimb of the brain injury during tailing, turning to the surgical side, scored 3 points; Grade IV is severe dysfunction, manifested as conscious change, lethargy, delayed response, Can not stand, i has 4 points; V is death, i has 5 points.
  • Model group Model of cervical-vein bypass model manufactured
  • Normal saline control group Equal volume injection of normal saline into the ear vein
  • Genetically engineered lysozyme low-dose group Intravenous vein injection gene Engineering fibrinolytic enzyme 1250U/kg
  • Genetically engineered plasmin middle dose group Intracerebral vein injection of genetically engineered plasmin 2500 U/kg
  • Genetically engineered plasmin high dose group Intracerebral vein injection of genetic engineering plasmin 5000U/ Kg
  • urokinase positive control group intravenous injection of urokinase 2 X 10 4 U/kg.
  • Method of administration After the establishment of the carotid-venous bypass model, the saline control group, the genetically engineered plasmin low-dose group, the middle-dose group, the high-dose group, and the urokinase-positive control group were administered 15 min after the bypass establishment. , observe 3h.
  • the low-dose, middle-dose and high-dose groups of genetically engineered plasminogen could significantly increase the FDP content compared with the model group, indicating that the genetically engineered plasmin can enhance the fibrinolytic activity of the blood system.
  • there was no significant change in FIB indicating that genetically engineered plasmin was less prone to bleeding side effects.
  • Drunk small dose group 0 7.8 4.9 0 2.5 5.34 ⁇ 2.80 ⁇
  • the ELT and FDP were counted by the number of cases. According to the rank sum test, compared with the saline group, both were p ⁇ 0.01; the FIB content was tested by q, ⁇ compared with the saline group, p ⁇ 0.05.
  • Relieving salt 8 85.31 ⁇ 3.61 28.25 + 1.02

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Abstract

La présente invention a trait à un gène enzymatique fibrinolytique de lombric, à des souches de génie génétique, et à leur construction et leurs utilisations. Ledit gène enzymatique fibrinolytique est constitué de 747 nucléosides codant pour une protéine de 246 acides aminés. Le gène peut être exprimé dans Pichia pastoris sous la forme d'une protéase fibrinolytique de génie génétique active. La présente invention a également trait à une variété d'applications de ladite protéase fibrinolytique de génie génétique pour le développement de médicaments dans des domaines thérapeutiques, de soins de santé et médicaux.
PCT/CN2005/000501 2004-04-14 2005-04-14 Gene enzymatique fibrinolytique de lombric, souches de genie genetique, et leur construction et leurs utilisations WO2005100570A1 (fr)

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Application Number Priority Date Filing Date Title
CN200410034475.9 2004-04-14
CN200410034474.4 2004-04-14
CN 200410034475 CN1563387A (zh) 2004-04-14 2004-04-14 一种蚯蚓纤溶酶基因和基因工程菌株及其构建和应用
CN 200410034474 CN1259411C (zh) 2004-04-14 2004-04-14 一种毕赤酵母基因工程菌株及制备方法和应用

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116500149A (zh) * 2023-03-07 2023-07-28 山东省食品药品检验研究院 一种蚓激酶的超高效液相色谱-串联质谱鉴别方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1057565C (zh) * 1998-06-11 2000-10-18 中国预防医学科学院病毒学研究所 正蚓科双胸蚓属蚯蚓纤溶酶基因核苷酸序列及其克隆方法
JP2002034566A (ja) * 2000-07-21 2002-02-05 Yokohama Kokusai Bio Kenkyusho:Kk ミミズ由来セリンプロテアーゼの遺伝子、当該遺伝子を含むプラスミドベクター及び形質転換体
KR100384467B1 (ko) * 2000-05-12 2003-05-22 주식회사 유젠바이오 한국산 지렁이에서 분리한 룸부로키나제 유전자 및 효모에서 룸부로키나제 단백질 대량 발현 방법

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1057565C (zh) * 1998-06-11 2000-10-18 中国预防医学科学院病毒学研究所 正蚓科双胸蚓属蚯蚓纤溶酶基因核苷酸序列及其克隆方法
KR100384467B1 (ko) * 2000-05-12 2003-05-22 주식회사 유젠바이오 한국산 지렁이에서 분리한 룸부로키나제 유전자 및 효모에서 룸부로키나제 단백질 대량 발현 방법
JP2002034566A (ja) * 2000-07-21 2002-02-05 Yokohama Kokusai Bio Kenkyusho:Kk ミミズ由来セリンプロテアーゼの遺伝子、当該遺伝子を含むプラスミドベクター及び形質転換体

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] 19 April 2003 (2003-04-19), Database accession no. (AY178854) *
DATABASE GENPEPT [online] 19 April 2003 (2003-04-19), Database accession no. (AAP04532) *
HU Y. ET AL: "Cloning and sequence analysis of earthworm fibrinolytic enzyme gene cDNA", J. WUHAN UNIV.(NAT.SCI.ED.), vol. 50, no. 2, 23 April 2004 (2004-04-23), pages 211 - 215 *
SUGIMOTO M. AND NAKAJIMA N.: "Molecular Cloning, Sequencing, and Expression of cDNA Encoding Serine Protease with Fibrinolytic Activity from Earthworm", BIOSCI. BIOTECHNOL. BIOCHEM., vol. 65, no. 7, July 2001 (2001-07-01), pages 1575 - 1580 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116500149A (zh) * 2023-03-07 2023-07-28 山东省食品药品检验研究院 一种蚓激酶的超高效液相色谱-串联质谱鉴别方法

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