WO2005100570A1 - Earthworm fribrinolytic enzyme gene, genetic engineering strains, and the construction and usages thereof - Google Patents

Earthworm fribrinolytic enzyme gene, genetic engineering strains, and the construction and usages thereof 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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Prior art keywords
plasmin
genetically engineered
protein
gene
pichia pastoris
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PCT/CN2005/000501
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French (fr)
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 200410034474 external-priority patent/CN1259411C/en
Priority claimed from CN 200410034475 external-priority patent/CN1563387A/en
Application filed by Green Life Laboratory Limited filed Critical Green Life Laboratory Limited
Publication of WO2005100570A1 publication Critical patent/WO2005100570A1/en

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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

The present invention discloses an earthworm fribrinolytic enzyme gene, genetic engineering stains, and the construction and usages thereof. Said fribrinolytic enzyme gene consists of 747 nucleosides, which encodes a protein of 246 amino acid. This gene can be expressed in Pichia pastoris into an active genetic engineering fribrinolytic protease. The present invention further discloses the wide applications of said genetic engineering fribrinolytic protease for drugs development in the areas of therapeutic, healthy, and medical cares.

Description

蚯蚓纤溶酶基因和基因工程菌株及其构建和应用 本申请要求 2004年 4月 14 日提交的申请号 200410034474.4 的 中 国专利申请和 2004 年 4 月 14 日 提交的申请号 200410034475.9的中国专利申请的优先权。 上述申请通过引证全 文并入本文。 发明领域 本发明涉及正蚓科粉正蚓属蚯蚓纤溶酶基因以及编码正蚓 科粉正蚓属丘蚓纤溶酶基因的核苷酸序列。 本发明还涉及正蚓科 粉正蚓属蚯蚓纤溶酶基因的制备方法。 本发明进一步涉及一种表 达该基因的毕赤酵母基因工程菌株的构建以及基因工程纤溶酶 在药物研究开发中的应用。 背景技术 急性心肌梗死、 脑梗死、 肺栓塞、 外周动月永血栓和深部静脉 血栓等心脑血管疾病是危害人类生命和健康的主要疾病之一。据 世界卫生组织统计， 全世界每年大约有 1200万人死于心脏病和 脑卒中。 在我国， 随着经济的高速发展， 人民群众物质生活水平 的提高，心脑血管疾病的发病率及其病死率亦正逐年提高。因此， 研制和发展高效、 特异、 安全、 不良反应小的溶血栓药物， 一直 是近年来世界范围的热门课题。 蚯蚓， 俗称地龙， 在我国已有数千年的应用历史， 可清热、 平肝、 止喘、 通络， 主治高热狂躁、 惊风抽搐等病症。 1983年曰 本 H.Mihara首次发现正蚓科 （Lumbrici dae)丘蚓水提物有直接溶 解纤维蛋白及纤溶酶原激活作用， 其活性成分称为纤溶酶 ( earthworm fibrinolytic enzyme, EFE ), 又称虫 激醉。 千溶醉溶 栓的药理机制主要有： 1)与纤维蛋白具有特殊亲和力，不仅水解富 含纤溶酶原的纤维蛋白， 还可以水解不含纤溶酶原的纤维蛋白， 使纤维蛋白迅速降解， 还可以水解纤维蛋白原。 2)类似 tPA, 激 活纤溶酶原而发挥间接作用， 体外实险表明纤溶酶对牛血管内皮 细胞 tPA的释放有刺激作用。 3 ) 水解凝血因子 I，抑制了血小板 在其表面的粘附。 研究结果表明： 蚯蚓体内的纤溶酶具有多种组分， 仅在粉正 蚓 （ Lumbricus mbellus ) 中已经分离到至少 6种不同的组分。 现 今国外已经从虫丘蚓中分离了 6个编码纤溶酶基因的 cDNA序列。 国内也已从赤子爱胜蚓、 双胸蚓等蚯蚓中克隆到了蚯蚓纤溶酶基 因， 但未见有从粉正蚓中分离纤溶酶基因并进行克隆表达的报 道。 目前已有多种蚯蚓纤溶酶制剂应用于临床， 主要为各种胶嚢 制剂， 都是从蚯蚓肠腔和体液中提取的， 如普恩复、 百奥、 博洛 克 ，溶栓胶囊等， 多用在心脑血管疾病方面。 此外由于纤 酶可 改善血液流变学， 降低血小板聚集率， 所以可以用在与血液流变 学相关的一些疾病中， 如美尼尔病、 突发性耳辈、 肺心病等。 传统的蚯蚓纤溶酶胶嚢制剂应用于临床有一定的疗效， 但也 存在明显的缺陷， 首先是成分复杂， 不能静脉注射， 而口服给药 作用緩慢， 因而不能应用于治疗急性心、 脑血管栓塞疾病； 其次 胶嚢制作工艺复杂， 生产受蚯蚓养殖季节、 周期的限制， 产品产 量、 质量不稳定； 从虫体中分离纯化纤溶酶工艺烦瑣， 产品中可 能含有各种小分子多肽或其它化学物质， 可能引起机体的过敏反 应。 而运用基因工程的方法则可以通过简单的发酵大量获得纤溶 酶， 分离纯化工艺简单， 产品純度高， 成本底。 因此， 基因工程 纤溶酶在医疗、卫生、保健类药物的研发领域中具有广泛的用途， 可用于制备用于治疗栓塞性心血管疾病的注射水剂、 口服药剂 等，还可用于生产具有预防血栓疾病、有益心血管健康的保健品。 在基因工程药物开发方面， 目前应用最广泛、 技术比较成熟 的表达系统有： 大肠杆菌系统、 酵母表达系统和 CHO细胞表达 系统。 大肠杆菌表达系统是目前应用最广泛、 最成熟的表达系统， 适合于表达非糖基化蛋白质及二级结构较简单的蛋白质， 表达产 物一般是在细胞质内形成包涵体， 产物需要经过复性才具有活 性， 也有的载体可分泌目的蛋白于细胞间质， 或在细胞质内可溶 性表达。 其最大的缺点是不能进行表达后的加工和修饰（比如糖 基化）， 而这种加工过程是很多蛋白质获得功能所必需的。 蚯蚓 plasmin gene and genetically engineered strain and construction and application thereof. The present application claims priority to Chinese patent application No. 200410034474.4 filed on Apr. 14, 2004, and the Chinese patent application No. 200410034475.9 filed on April 14, 2004. right. The above application is incorporated herein by reference in its entirety. FIELD OF THE INVENTION The present invention relates to a nucleotide sequence of the genus 蚯蚓 蚯蚓 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. BACKGROUND OF THE INVENTION 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. therefore, The development and development of high-efficiency, specific, safe, and adverse reactions to thrombolytic drugs has been a hot topic in the world in recent years.蚯蚓, commonly known as the earth dragon, has been used in China for thousands of years, can clear heat, calm the liver, stop asthma, collaterals, attending high fever, convulsions and convulsions. In 1983, H. Mihara first discovered that the water extract of the genus Lumbrici dae has direct activation of fibrin and plasminogen activation. The active ingredient is called earthworm fibrinolytic enzyme (EPE). Also known as insects drunk. 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. The results showed that: 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. In China, 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. At present, a variety of 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. In addition, since 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. First, 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; Secondly, 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. Therefore, 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. In the field of genetic engineering drug development, the most widely used and mature technology expression systems are: Escherichia coli system, yeast expression system and CHO cell expression system. 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细胞表达系统:这是目前构建及表达难度相对较大的表 达系统， 适合表达糖基化蛋白和空间结构较复杂的蛋白质， 表达 产物一般存在于细胞质中， 如果在基因构建时在目的蛋白前加一 段信号肽， 表达产物会分泌到胞外， 通过细胞翻译后包装成为有 活性的蛋白， 但表达量低、 成本高、 纯化困难使其难以在工业化 生产中应用。 因此， 在 蚓纤溶酶的发展应用中， 需要一种表达量高， 稳 定性好， 活性高， 能进行蛋白质翻译后加工， 便于工业化生产的 表达系统。 发明概述 本发明的目的之一在于提供一种正蚓科粉正蚓属虫丘蚓纤溶 酶基因以及其核苷酸序列 SEQ ID NO: 1。 本发明的目的之一在于提供一种正蚯蚓粉正蚓属蚯蚓纤溶 酶基因编码的丘蚓纤溶酶蛋白质。 ά丘蚓纤溶酶蛋白质包含 SEQ ID NO: 2的氨基酸序列。 本发明的目的之一在于提供一种制备正蚓科粉正蚓属蚯蚓 纤溶酶基因的方法。 本发明的目的之一在于提供一种基因工程菌株， 该菌株能表 达蚯蚓纤溶酶。 本发明还涉及该菌株的制备方法， 用于制备基因 工程纤溶酶蛋白。 本发明的目的之一在于提供了一种正蚓科粉正蚓属蚯蚓纤 溶酶基因的制备及克隆到大肠杆菌 （E.coli ) 的方法。 本发明的目的之一提供构建基因工程毕赤酵母菌株的方法， 构建的基因工程菌株可以用来表达蚯蚓纤溶酶基因， 获得基因工 程纤溶酶蛋白。 本发明的目 的之一是提供一种基因工程毕赤酵母菌株 Pichia pastoris X-33 (Ppic6 a A-EFE)。 本发明的目 的之一是提供一种基因工程毕赤酵母菌株 Pichia pastoris X-33 (Ppic6 A-EFE)。 本发明的另一个目的在于提供一种治疗心脑血管疾病药物， 其包含基因工程蚯蚓纤溶酶蛋白。 本发明的另一个目的在于提供一种治疗心血管疾病药物， 其 包含基因工程蛭蚓纤溶酶蛋白和药学上可接受的载体。 本发明的另一个目的在于提供一种与血液流变学相关的疾 病的药物， 其特征在于所述药物包含基因工程 蚓纤溶酶蛋白。 本发明的另一个目的在于提供一种与血液流变学相关的疾 病的药物， 其特征在于所述药物包含基因工程丘蚓纤溶酶蛋白和 药学上可接受的载体。 本发明的另一个目的是提供治疗栓塞性心脑血管疾病的药 剂， 其特征在于所述药剂包含基因工程 蚓纤溶酶蛋白。 本发明的另一个目的是提供治疗栓塞性心脑血管疾病的药 剂， 其特征在于所述药剂包含基因工程蚯蚓纤溶酶蛋白和药学上 可接受的载体。 本发明的另一个目的在于提供预防血栓疾病有益心血管健 康的保健品。 本发明的另一个目的是提供一种基因工程蚯蚓纤溶酶蛋白 在制备治疗心脑血管疾病药物中的应用。 附图说明 本发明的目的， 特点和优势可通过对本发明的详细描述和附 图演示。 附图用来进一步说明演示本发明， 不解释为对,本发明的 任何限制。 附图 1 纤溶酶基因的 PCR扩增结果。 泳道 1为 DNA分子量标准 （ DNA/Hindlll ); 泳道 2为反 转录合成的 cDNA第一条链； 泳道 3为纤溶酶基因的 PCR扩增 产物 附图 2 重组质粒 pUCm-T-EFE的酶切鉴定结果。 泳道 1 为 DNA分子量标准 （ DNA/ Hind III ); 泳道 2、 3 钎溶酶基因的 PCR扩增产物， 泳道 4为 DNA分子量标准 （ λ DNA/EcoR 1+ Hind III );泳道 5为重组质粒 pUCm-T-EFE用 Not I 和 Sal I双酶切的结果； 泳道 6为重组质粒 pUCm-T-EFE用 Sal I 单酶切的结果； 泳道 7为重组质粒 pUCm-T-EFE用 Sph I单酶切 的结果。 附图 3 重组表达载体 pPIC6 oc A-EFE和 pPIC6A-EFE的酶切 鉴定结果。 泳道 1为 PCR分子量标准； 泳道 2为重组表达载体的 PCR 鉴定结果；泳道 3为重组表达载体用 EcoR I+Xba I双酶切的结果； 泳 i£ 4为重组表达载体用 Sac I单酶切的结果；泳道 5为 DNA分 子量标准 （ DNA/EcoR I+ Hind III )。 附图 4基因工程纤溶酶纯化后的 SDS - PAGE电泳检测结果。 泳道 1为小分子量蛋白标准； 泳道 2为纯化后的基因工程菌 Pichia pastoris X-33 ( pPIC6 a A-EFE)上清， 即基因工程纤溶酶蛋 白。 附图 5 纤维蛋白平板法测定纤溶酶的生物学活性。 a为纯化后的基因工程纤溶酶蛋白； b为 lOul纤溶酶标准品 (10000U/ml); c为 lOul对照菌 Pichia pastoris X-33(pPIC6 α Α)上 清纯化后发酵产物。 附图 6 纤维蛋白平板法测定纤溶酶的生物学活性 a为基因工程纤溶酶蛋白法发酵上清液； b为 lOul纤溶酶标 准品（10000U/ml); c为 lOul对照菌 pichia pastoris X-33(pPIC6A) 发酵产物。 发明详述 本发明的优选实施方案描述如下。 本发明提供了一种正蚓科粉正蚓属蚯蚓纤溶酶基因的核苷 酸序列及其制备方法。 在本发明的一个实施例中提供了一种正蚓科粉正蚓属 ά丘蚓 纤溶酶基因的核苷酸序列及其制备方法， 该方法是以正蚓科粉正 蚓属蚯蚓总 RNA为模板，用特异性寡核苷酸引物，经反转录 PCR 方法得到纤溶酶基因 cDNA。 粉正蚓购于华中农业大学， 基因测 序结果表明 cDNA由 747核苷酸组成， 5 '端至 3 '端序列为： 正蚓科粉正蚓属蚯蚓纤溶酶基因的核苷酸序列 （SEQ ID ΝΟ: 1 ) acatggaacttcctcccggaacaaaaattgtcggaggaattgaagctagaccatacgagttccca tggcaggtgtccgtccgaaggaaatcttccgattcccatttctgcggaggtagcatcatcaacgat cgttgggttgtctgcgctgctcactgcatgcagggagaggcccccgctctggtttcattggtcgtg ggtgagcacgacaggagtgcagcgagtacagtacgtcagactcatgacgttgatagcatcttcg ttcacgaggactacaacacaaataccctagagaacgacgtttctgtcatcaagacatctgttgcca tcactttcgacatcaacgttggtccaatctgcgccccagatccggctaacgactacgtctaccgta agagccagtgttccggatggggaactatcaattcaggtggaatctgctgtcccaacgttctgcgat acgtgacgctgaatgacacaaccaaccaatactgcgaagatgtatacccactaaattcaatctac gacgatatgatttgcgcgtcggacaacactgggggtaacgacagagactcctgccagggtgact ccggcggccctctgagcgtcaaggatggtagtggaatcttcagcctgattggtattgtgtcttggg gaattggttgcgcttctggctatccaggagtctactcccgcgtcggattccatgctgcatggatca ccgacatcatcaccaacaactaaaccg 其中 3-740位核苷酸是基因编码的成熟肽序列， 即纤溶酶蛋 白序列， 741-743位是终止密码子 TAA。 该基因编码的蛋白质氨 基酸序列为： 正蚓科粉正蚓属蚯蚓纤溶酶的氨基酸序列 （ SEQ ID NO:2 )
Figure imgf000012_0001
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. SUMMARY OF THE INVENTION 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 蚯蚓 蚯蚓 蚓 蚯蚓 蚯蚓 plasmin gene. The ά 蚓 蚓 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). One of the objects of the present invention is to provide a genetically engineered Pichia pastoris strain Pichia pastoris X-33 (Ppic6 A-EFE). Another object of the present invention is to provide a medicament for treating cardiovascular and cerebrovascular diseases comprising genetically engineered 蚯蚓 plasmin protein. Another object of the present invention is to provide a medicament for treating cardiovascular diseases comprising a genetically engineered 蛭蚓 plasmin protein and a pharmaceutically acceptable carrier. 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 蚓 plasmin protein. 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 蚓 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 蚯蚓 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. Another object of the present invention is to provide a genetically engineered plasmin protein for use in the preparation of a medicament for treating cardiovascular and cerebrovascular diseases. BRIEF DESCRIPTION OF THE DRAWINGS The objects, features, and advantages of the present invention will be apparent from the description and appended claims. The drawings are intended to further illustrate the invention and are not to be construed as limiting the invention. Figure 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. Figure 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 (λ 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 μl of the control Pichia pastoris X-33 (pPIC6 α Α) 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. In one embodiment of the present invention, a nucleotide sequence of a scorpion scorpion plasmin gene and a preparation method thereof are provided, and the method is a genus of genus As a template, 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 results of gene sequencing indicated that the cDNA consisted of 747 nucleotides, and the 5' to 3' end sequence was: the nucleotide sequence of the 蚯蚓 plasmin gene of the genus ID ΝΟ: 1) acatggaacttcctcccggaacaaaaattgtcggaggaattgaagctagaccatacgagttccca tggcaggtgtccgtccgaaggaaatcttccgattcccatttctgcggaggtagcatcatcaacgat cgttgggttgtctgcgctgctcactgcatgcagggagaggcccccgctctggtttcattggtcgtg ggtgagcacgacaggagtgcagcgagtacagtacgtcagactcatgacgttgatagcatcttcg ttcacgaggactacaacacaaataccctagagaacgacgtttctgtcatcaagacatctgttgcca tcactttcgacatcaacgttggtccaatctgcgccccagatccggctaacgactacgtctaccgta agagccagtgttccggatggggaactatcaattcaggtggaatctgctgtcccaacgttctgcgat acgtgacgctgaatgacacaaccaaccaatactgcgaagatgtatacccactaaattcaatctac gacgatatgatttgcgcgtcggacaacactgggggtaacgacagagactcctgccagggtgact ccggcggccctctgagcgtcaaggatggtagtggaatcttcagcctgattggtattgtgtcttggg gaattggttgcgcttctggctatccaggagtctactcccgcgtcggattccatgctgcatggatca ccgacatcatcaccaacaactaaaccg wherein 3-740 nucleotide sequence of the gene encoding the mature peptide, i.e., plasmin protein sequence, stop codon position 741-743 TAA. The amino acid sequence of the protein encoded by the gene is: the amino acid sequence of the genus Fibrinolytic enzyme of the genus Orthodox genus (SEQ ID NO: 2)
Figure imgf000012_0001
FVHEDYNTNTLENDVSVIKTSVAITFDINVGPICAPDPANDYVY
Figure imgf000012_0002
FVHEDYNTNTLENDVSVIKTSVAITFDINVGPICAPDPANDYVY
Figure imgf000012_0002
SWGIGCASGYPGVYSRVGFHAAWITDIITNN 本发明涉及的纤溶酶蛋白分子量为 26.4kD, 等电点为 4.5， 富含甘氨酸、丝氨酸、缬氨酸和天冬氨酸。其生物化学特性还有：SWGIGCASGYPGVYSRVGFHAAWITDIITNN 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:
①热稳定性： 纤溶酶 60°C加热 30分钟后， 酶活性没有明显降低 ， 65 °C以上，酶活性开始下降 ， 85 °C加热 30 分钟酶活性完全丧失;1 Thermal stability: Fibrinolytic enzyme After 60 minutes of heating at 60 °C, the enzyme activity did not decrease significantly. Above 65 °C, the enzyme activity began to decrease, and the enzyme activity was completely lost after heating at 85 °C for 30 minutes.
②酸稳定性：纤溶酶在 pHl ~ 11间酶活性没有明显变化，ρΗ低于 1或高于 11 时， 酶活性明显下降， 最佳纤溶反应的 ρΗ值范围较 宽， 在 7 ~ 10之间。 本发明涉及的纤溶酶基因还可以通过以下方法获得： （ 1 )亚 克隆： 以本发明所涉及的基因片段为模板， 或者以含有本发明所 涉及的基因片段的重组质粒为模板， 或者以含有本发明所涉及的 基因片段的基因工程菌株 DNA为模板， 通过常规分子生物学操 作方法 （如 PCR ), 亚克隆得到相应基因片段， 这些片段可以包 括全部的或者部分的本发明所涉及基因序列， 这些片段可在细 菌、 酵母、 动植物细胞或其它真核、 原核生物中表达， 得到蛋白 质或多肽片段， 这些蛋白质或多肽片段与本发明所涉及的蛋白质 有相同的生物学功能； （2 )人工合成： 根据本发明所涉及的氨基 酸或者核苷酸序列， 可以通过化学合成的方法合成 DNA片段， 以克隆到相应的载体中进行表达， 表达的蛋白质或多肽与本发明 所涉及的蛋白质有相同的生物学功能。 本发明所涉及的基因片段可以进行修饰， 比如突变基因的某 些核苷酸序列， 但并不影响其编码的蛋白质氨基酸序列； 可以增 加、 缺失本发明所涉及的氨基酸或者核苷酸序列， 这些修饰不会 影响蛋白质的生物化学特性、 高级结构和蛋白质的生物学功能， 比如： 可以在蛋白质或多肽 N-端或 C-端加上分泌信号肽， 还可 以加上适当的接头 （如 6个组氨酸）便于蛋白质提取、 纯化。 本发明所涉及的基因与载体连接后可以克隆到相应的宿主 中， 以便于基因的保存、 扩增和表达。 应用于本发明的克隆载体 包括但不仅限于下列载体： pBR322、 pBR325、pACYC177、pUC8、 pUC9、 pUC18、 pUC19> pLG339, pR290、 pKC37、 pKClOK SV 40、 pBluescript II SK +/- 、 pQE, pGEM-T、 pET系列载体。 载体特征及克隆方法可参见冷泉港实验室编写的《分子克隆实猃 手册》， 其内容并入本文作为参考。 应用于本发明的宿主——载 体系统包括但不仅限于下列系统： 细菌——噬菌体系统， 细菌一 一质粒载体系统， 酵母——酵母载体系统， 哺乳动物——病毒系 统 （牛痘病毒、 腺病毒等）， 昆虫细胞——杆 ^！犬病毒系统。 在本发明的一个实施例中提供了一种正蚓科粉正蚓属蚯蚓 纤溶酶基因的制备及克隆到大肠杆菌 （E.coli ) 的方法， 该方法 包括： 扩增引物合成， 蚯蚓总 RNA提取， mRNA的反转录， 目 的基因的 PCR扩增、 目的基因片段的回收、 目的基因片段克隆 入载体、 丘蚓纤溶酵 cDNA序列测定。 2 Acid stability: There was no significant change in enzyme activity between pH1 and 11 in plasmin. When ρΗ was lower than 1 or higher than 11, the enzyme activity decreased significantly, and the optimal fibrinolytic reaction ranged from ρΗ to 7-10. between. 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. These 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; (2) 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. In one embodiment of the present invention, there is provided a method for preparing a sputum sputum plasmin gene and cloning into Escherichia coli (E. coli), the method comprising: amplifying primer synthesis, 蚯蚓 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.
( 1 )扩增引物合成： 根据 GeneBank中已经发表的丘蚓纤溶 酶 cDNA序列设计 PCR寡核苷酸扩增引物。 上游引物 （P1 ) 长 18 个核苷酸： ACATGGAACTTCCTCCCG , 下游引物 （ P2 ) 长 19个核苷酸： ATC ACC AAC AACTAAACCG ,引物经 PACE纯化。 (1) 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, and the downstream primer (P2) is 19 nucleotides long: ATC ACC AAC AACTAAACCG, and the primers are purified by PACE.
( 2 )蚯蚓总 RNA提取： 取 3〜5条蚯蚓成体， 用 DEPC处理 过的灭菌水漂洗后， 放在铺有湿滤纸的平亚中饥饿过夜， 使其尽 可能的吐尽体内的泥沙。 再次用 DEPC水清洗干净后， 在液氮中 研磨成粉末状， 按照 QIAGEN 公司 RNeasyR Mini Kit的产品说 明书提取总 RNA。 将提取到的 RNA溶液贮存于 -80°C以备用。 (2) Extraction of total RNA from sputum: Take 3~5 sputum adults, rinse with sterilized water treated with DEPC, and starve overnight in Pingya, which is coated with wet filter paper, so as to spit out the mud in the body as much as possible. sand. After washing again with DEPC water, it was ground to a powder form in liquid nitrogen, and total RNA was extracted according to the product specification of QIAGEN RNeasyR Mini Kit. The extracted RNA solution was stored at -80 ° C for use.
( 3 )反转录合成 cDNA 第一条链： 按照 Promega 公司的 Reverse Transcription Reaction试剂盒说明书进行，以 Oligo(dT)20 为引物合成 cDNA第一链。 反应条件为： 42°C 1 小时； 95 °C 5 分钟； 3 °C 5分钟。 (3) Reverse transcription synthesis of cDNA The first strand: 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.
( 4 ) 目的基因的 PCR 扩增： 按照 Reverse Transcription Reaction试剂盒中给出的 PCR反应体系参数进行 PCR反应。 以 cDNA第一条链为模板进行 PCR扩增， 其反应参数为： 95 °C预变 性 4分钟； 94°C变性 1分钟， 55 °C退火 1.5分钟， 72°C延伸 2分 钟， 20个循环； 94°C变性 1分钟， 55 °C退火 1.5分钟， 72°C延 伸 2分钟， 10个循环； 72°C再延伸 10分钟。 (4) PCR amplification of the target gene: 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.
( 5 )目的基因片段的回收：按照上海华舜生物工程公司 PCR 产物回收试剂盒说明书回收目的基因片段。 (5) Recovery of the target gene fragment: The target gene fragment was recovered according to the instructions of the PCR product recovery kit of Shanghai Huasheng Bioengineering Co., Ltd.
( 6 ) 目的基因片段克隆入载体： 回收的 PCR产物与 T载体 连接， 连接产物转化感受态细胞大肠杆菌 ( E.coli ) JM109, 挑取 菌落快速提取质粒进行酶切鉴定， 所得到的阳性克隆子是包含本 发明涉及基因的大肠杆菌， 用于基因的增殖和保藏。 (6) 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.
( 7 ) 丘蚓纤溶峰 cDNA序列测定 提耳又质粒 DNA双脱氧法测定核苷酸序列， 测序引物为 M13 启动子引物。 正蚓科粉正蚓属蚯蚓纤溶酶基因核苷酸的序列测定 结果：蚯蚓纤溶酶 cDNA序列长 747个核苷酸，腺嘌呤核苷（A )、 包嘧啶核苷 （C )、 鸟嘌噙核苷 （G ) 及胸腺嘧啶核苷 （T ) 的含 量分别为： A-185、 C-197、 G-189; T-176。 基因成熟肽序列为第 3-740位核苷酸， 编码 246个氨基酸， 其余核苷序列为非编码区。 该蛋白质 cDNA成熟肽终止密码子位于基因第 741-743位， 终止 密码为 TAA。 本发明提供了表达纤溶酶基因的毕赤酵母基因工程菌株的 构建及表达， 该菌株可以表达纤溶酶基因， 得到基因工程纤溶酶 蛋白用于制备治疗心血管疾病的药物以及预防血栓疾病有益心 血管健康的保健品。 本发明所涉及的纤溶酶基因可以在包括但不仅限于下列载 体-宿主系统中进行表达， 获得基因工程蛋白： 大肠杆菌系统、 酵母表达系统、 CHO 细月包表达系统、 杆状病毒表达系统、 丝状 真菌表达系统、 芽孢杆菌表达系统。 毕赤 （Pichia ) 酵母表达系统是近年来发展起来的一种高效 的真核表达系统， 与其它表达系统相比， 它具有以下优点： （ 1 ) 表达量高， 因为毕赤酵母生长速度快， 而且表达载体使用的是强 启动子——乙醇氧化酶启动子， 外源蛋白表达量很高。 （2 )稳定 性好， 由于该系统的表达载体在酵母中不是以自主复制的质粒存 在， 而是通过同源重组稳定的整合在酵母染色体上， 所以构建的 工程菌株十分稳定。 ( 3 ) 纯化容易， 操作简便。 表达产物一般分 泌到胞外， 可以直接从培养液中检测目的蛋白， 不需要复性， 而 且可以直接从上清中提取蛋白。 （4 )活性高。 外源蛋白在毕赤酵 母中分泌表达的最大好处就是能够进行蛋白质翻译后加工， 包括 二硫键的成形成、 蛋白质折叠及糖基化等。 与啤酒酵母等其他一 些真核表达系统相比， 毕赤酵母对外源蛋白糖基化更接近于哺乳 动物的糖基化， 因此毕赤酵母表达的糖蛋白被认为更适宜用于人 体。 （5 )便于工业化生产。 毕赤酵母有现成的、 成熟的发酵技术 进行扩大培养， 而且毕赤酵母表达系统可以利用甲醇作为唯一碳 源生长， 大规模发酵生产方便， 成本低， 易于实现产业化。 本发明涉及的丘蚓纤溶酶 cDNA 来源于粉正蚓 （Lumbricus rubellus ), 该片段共有 741bp， 编码 246个氨基酸。 本发明在酵 母系统中表达该基因， 通过发酵的方法生产得到基因工程纤溶 酶， 并通过纤维蛋白平板法检测了该蛋白的生物活性。 目前国内 已有蚯蚓纤溶酶在大肠杆菌中表达方面的研究， 但其在酵母中的 表达未见艮道。 毕赤酵母表达载体包括但不仅限于下列载体： pPIC 6、 pPIC 6 oc、 pPIC Z、 pPIC Z, pPIC9k、 pPIC3.5k、 pPA0815、 pGAPZ, pGAP a Z;毕赤酵母表达菌株包括但不仅限于下列菌林： GS115、 KM71、 KM71H、 X-33、 SMD1168、 SMD1168H。 在本发明的一个实施例中提供了一种构建基因工程菌株、 在 毕赤酵母中表达蚯蚓纤溶酶基因的方法， 该方法是用 pPIC 6 c A 载体（Invitrogen公司）在毕赤酵母 X-33中表达蚯蚓纤溶酶基因， 毕赤酵母 Pichia pastoris X-33 ( pPIC6 a A-EFE ), 该菌株在中国 典型培养物保藏中心 （CCTCC )保藏， 编号为： M204005 , 该菌 株的制备方法为 （ 1 )蚯蚓纤溶酶基因的 PCR扩增； （2 ) PCR产 物克隆至 T载体； （3 )穿梭表达质粒的构建； （4 )表达纤溶酶基 因的酵母基因工程菌株的构建， 其步骤如下： ( 1 ) 蚯蚓纤溶酶基因的 PCR扩增： 以含有纤溶酶基因的 T 载体质粒为模板，才艮据 EFE成熟肽和表达载体 pPIC6 a A上的克 隆位点设计引物， 并在上游引物中加入 6个连续的组氨酸编码序 列， 之后加入肠激酶切割位点序列， 并设计了一对丘蚓纤溶酶 PCR扩增引物。 因此， 该工程菌株的表达户物为融合蛋白， 在纤 溶酶蛋白的 N端有 6个连续的組氨酸，用于表达产物的分离纯化， 纯化产物经过肠激酶切割后可切去融合蛋白 N 端的非天然存在. 氨基酸， 得到与天然纤溶酶组成完全相同的基因工程蛋白。 在本 发明的一个实施例中提供了一对引物序列 , 上游引物 P1 : (7) Determination of the nucleotide sequence of the scorpion fibrinolytic peak cDNA sequence and the plasmid DNA dideoxy method. The sequencing primer was the M13 promoter primer. Sequence analysis of nucleotides of 蚯蚓 蚯蚓 蚯蚓 蚯蚓 蚯蚓 蚯蚓 蚯蚓 蚯蚓 蚯蚓 蚯蚓 蚯蚓 蚯蚓 蚯蚓 蚯蚓 蚯蚓 蚯蚓 cDNA cDNA cDNA cDNA 747 747 747 747 747 747 747 747 747 747 747 747 747 747 747 747 747 747 747 747 747 747 747 747 747 The contents of purine nucleoside (G) and thymidine (T) are: A-185, C-197, G-189; T-176. 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. Since the expression vector of the system is not present in the yeast by the autonomously replicating plasmid, but is stably integrated on the yeast chromosome by homologous recombination, 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. Compared with other eukaryotic expression systems such as brewer's yeast, 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. At present, there is research on the expression of 蚯蚓 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. In one embodiment of the present invention, there is provided a method of constructing a genetically engineered strain for expressing a sputum plasmin gene in Pichia pastoris using a pPIC 6 c A vector (Invitrogen) in Pichia pastoris X- 33 expressed 蚯蚓 plasmin gene, Pichia pastoris X-33 ( pPIC6 a A-EFE ), the strain was deposited in the China Center for Type Culture Collection (CCTCC), number: M204005, the preparation method of the strain is (1) PCR amplification of 蚯蚓plasmin gene; (2) PCR product cloning into T vector; (3) Construction of shuttle expression plasmid; (4) Construction of yeast genetically engineered strain expressing plasmin gene, steps as follows: (1) PCR amplification of 蚯蚓plasmin gene: The T-vector plasmid containing the plasmin gene was used as a template to design primers based on the cloning site of the EFE mature peptide and the expression vector pPIC6 a A, and upstream primers. Six consecutive histidine coding sequences were added, followed by the enterokinase cleavage site sequence, and a pair of 蚓 蚓 plasmin PCR amplification primers were designed. Therefore, the expression product of the engineered strain is a fusion protein, and there are 6 consecutive histidines at the N-terminus of the plasmin protein for separation and purification of the expression product, and the purified product can be excised by the enterokinase cleavage. The non-naturally occurring N-terminus. Amino acid, which has the same genetically engineered protein as the natural plasmin. In one embodiment of the invention a pair of primer sequences is provided, upstream primer P1:
CGAATTCCACCACCACCACCACCACGGTGGTGGTAGCA GCGACGACAAG ATGGAACTTCCTCCCGGAACA (下划线处 为 内 切 酶 EcoR I 位 点 ） 。 下 游 引 物 P2 ： GTCTAGATTAGTTGTTGGTGATGATGTCGGTGATCCATGCAG CAT (下划线处为内切酶 Xba l位点：)。 CGAATTCCACCACCACCACCACCACGGTGGTGGTAGCA GCGACGACAAG ATGGAACTTCCTCCCGGAACA (underlined is the endonuclease EcoR I site). The downstream primer P2: GTCTAGATTAGTTGTTGGTGATGATGTCGGTGATCCATGCAG CAT (underlined is the endonuclease Xba l site:).
( 2 ) PCR产物克隆至 T载体：回收 PCR产物中的基因片段， 并克隆于 pGEM-T载体， 将该重组载体（ pGEM-T-EFE2 )转入大 肠杆菌 JM109中。 (2) 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.
( 3 ) 穿梭表达质粒的构建： 提取重组载体 pGEM-T-EFE2， 用 EcoR I 和 Xba I双酶切以得到目的片段 EFE; 同样酶切 pPIC6 α A空载体并在 CIAP作用下使之去磷酸化。 将去磷酸化后的线 性化的 pPIC6 a A空载体与双酶切后得到的 EFE 在 T4DNA连接 酶的作用下 4Ό过夜， 以得到重组表达载体 pPIC6aA-EFE, 将 该重組载体转入大肠杆菌 JM109中进行扩增。 (3) Construction of shuttle expression plasmid: The recombinant vector pGEM-T-EFE2 was extracted and digested with EcoR I and Xba I to obtain the target fragment EFE; the pPIC6 α A empty vector was also digested and dephosphorylated under the action of CIAP. Chemical. Dephosphorylated line The recombinant pPIC6 a A vector and the double-cut EFE were incubated overnight under the action of T4 DNA ligase to obtain the recombinant expression vector pPIC6aA-EFE, which was transformed into E. coli JM109 for amplification.
(4)表达纤溶酶基因的酵母基因工程菌株的构建； a. 穿梭表达质粒的线形化： 提取重组质粒 pPIC6ocA-EFE， 用 Sacl酶切成线性， 同样酶切空质粒 pPIC6aA作为对照。 b. 转化： 方法参考 Invitrogen公司表达载体说明书： 利用生 化方法将线性化的重组质粒 pPIC6o A-EFE和空质粒 pPIC6o A 转化 X-33酵母菌株,在含有杀稻瘟素 （ Blasticidin ) 300mg/ml的 YPD ( 1% 酵母抽提物、 2% 蛋白胨、 2%葡萄糖） 固体培养基上 培养 2-3天， 直到有单菌落出现。 c筛选阳性转化子： 4兆取 10-20个单菌落于 YPD液体培养基 中 30°C, 250rpm摇床培养直至对数生长晚期， 提取基因组进行 重组子的 PCR鉴定。 引物为 Invitrogen公司表达载体提供的通用 引物， 其序列为上游： GACTGGTTCCAATTGAC AAGC , 下游： GCAAATGGCATTC TGACATCC,鉴定的阳性克隆毕赤酵母工程 菌株命名为 Pichia pastoris X-33 ( pPIC6 a A-EFE ), 该菌株在中 国典型培养物保藏中心保藏编号为： CCTCC M204005。 在毕赤酵母中有两个编码乙醇氧化酶的基因 AOX1 和 AOX2 , 可以利用曱醇作为唯一碳源快速生长， 而当培养基中存 在其它碳源时， AOX 基因不表达。 在毕赤酵母工程菌株中， 外 源基因被克隆至 AOX1启动子控制下， 因此必须以曱醇作为诱导 剂， 同时也是唯一碳源来实现外源蛋白的高效表达， 在诱导表达 时， 应补充甲醇以维持其 0.5%的含量。 在本发明的一个实施例中提供了一种毕赤酵母工程菌株诱 导表达的方法， 可用于毕赤酵母工程菌株诱导表达的培养基包括 但不仅限于下列培养基： MGY、 MM、 BMM、 BMMY, 各种培 养基的配置方法为本技术领域人员非常熟悉的方法， 可以通过参 考相关文献获得详情。 蛋白质的分离和纯化有多种方法， 其原理是根据目的蛋白的 物理和化学性质将其与其他蛋白质分离。 如根据蛋白质分子量的 大小和蛋白形状可选择离心法、 超滤法、 凝胶过滤层析法， 根据 蛋白盾溶解度的不同可选择硫酸铵沉淀法或丙酮沉淀法， 根据等 电点的不同选择等电聚焦电泳法或等电沉淀法， 才艮据疏水性不同 可选择疏水作用层析法、 反向 HPLC法， 根据蛋白质与金属离子 的结合能力选择固定化金属亲和层析法。 在本发明的一个实施例中提供了一种在毕赤酵母 X-33 中表 达、 分离、 纯化蚯蚓纤溶酶基因， 获得基因工程蛋白的方法， 该 方法包括： （ 1 )毕赤酵母工程菌株的诱导表达； （2 )基因工程纤 溶酶的分离、 纯化； （3 )基因工程纤溶酶蛋白的功能研究及活性 测定。 (4) Construction of yeast genetically engineered strain expressing plasmin gene; a. Linearization of shuttle expression plasmid: The recombinant plasmid pPIC6ocA-EFE was extracted, and the plasmid was cut into linear by Sacl, and the empty plasmid pPIC6aA was also digested as a control. b. Transformation: Method Reference Invitrogen Expression Vector Specification: The linearized recombinant plasmid pPIC6o A-EFE and the empty plasmid pPIC6o A were transformed into X-33 yeast strain by biochemical method, containing blasticidin (Blastidin) 300 mg/ml. YPD (1% yeast extract, 2% peptone, 2% glucose) was incubated on solid medium for 2-3 days until a single colony appeared. c Screening for positive transformants: 4 megagrams of 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 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. In 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. In the Pichia pastoris engineering strain, 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%. In one embodiment of the present invention, a method for inducing expression of a Pichia pastoris engineering strain is provided, and the medium which can be used for inducing expression of Pichia pastoris engineering strains 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. There are several methods for the 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. According to the solubility of the protein shield, 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. In one embodiment of the present invention, a method for expressing, isolating, and purifying a 蚯蚓 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.
( 1 ) 毕赤酵母工程菌林的诱导表达 毕赤酵母工程菌株诱导表达方法如下： 挑取重组菌的单菌落 到 20mlYPD液体培养基， 30°C、 250rpm培养进行活化； 16个小 时后取 10ml 活化后的菌液转接 500ml MGY液体培养基 30 °C、 250rpm培养增浓；增浓 24小时后的菌液经离心收集菌体（ 1500g、 10分钟、 4 °C ); 将收集到的菌体用重悬于 1000ml MM液体培 养基进行 i秀导表达， 每 24h添加曱醇至终浓度为 0.5%; i秀导 96h 后离心收集发酵液上清 （ 30000g、 20分钟、 4 °C ); (1) Induction of 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);
( 2 )基因工程纤溶酶的分离、 纯化 可用于基因工程纤溶酶分离、 純化的方法包括但不仅限于下 列方法： Ni2+柱层析法、 离子交换层析法、 亲和层析法、 凝胶过 滤层析法、 电泳回收法、 超滤法、 u酸铵沉淀法等。 在本发明的实施例中提供一种分离、 纯化基因工程纤溶酶的 方法， 分为 3个步骤， 首先是 Ni2+柱层析纯化， 基因工程纤溶酶 蛋白有 6个连续的组氨酸残基，它们能与 Ni2+结合使基因工程蛋 白结合在固定的金属离子柱上， 而其它杂蛋白不能与金属离子柱 结合被直接洗脱下来， 最后用适当的洗脱液将目的蛋白质洗脱下 来从而达到蛋白纯化的目的。 Ni2+柱层析纯化产物加入高度纯化 的牛内肠激酶或重组的内肠激酶， 进行降解反应， 可切去融合蛋 白中的非天然氨基酸残基， 得到与天然蛋白氨基酸组成完全相同 的基因工程蛋白 。 最后将肠激酶降解的样品直接加入 QAE-Tyoypearl 550C层析柱中， 由于不同蛋白质等电点的不同将 基因工程纤溶酶分离纯化， 纯化产物经 SDS - PAGE电泳分析验 证。 (2) Isolation and purification of genetically engineered plasmin. 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. In the embodiment of the present invention, 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. Base, they bind to Ni2+ to bind genetically engineered proteins to a fixed metal ion column, while other heteroproteins cannot be combined with metal ion columns. The binding is eluted directly, and finally the protein of interest is eluted with an appropriate eluent to achieve protein purification. The purified product of 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. . Finally, the 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.
( 3 )基因工程纤溶酶蛋白的功能研究及活性测定 当前尚无丘蚓纤溶酶活性测定的国家标准， 然而作为一种新 的药品， 需要建立一套活性和效价测定的方法。 同时这种测定方 法应该既准确可靠， 又简便易行。 当前的测定方法只能参考其它 溶栓药物的活性测定方法。 比如尿激酶活性的气泡上升法和测定 蝮蛇抗栓酶的精氨酸脂酶法 （TAME ) 法， 但这两种方法均不适 用测定蚯蚓纤溶酶， 因为纤溶酶具有纤维蛋白溶酶原激活剂和蛋 白水解酶的作用， 在溶解血栓时能发挥这两种功能， 而且该酶的 溶酶活性显著大于激酶活性。 纤维蛋白平板法能够客观地表示出纤溶酶溶栓的能力。 血纤 维蛋白平板中含有纤维蛋白溶酶原， 在纤溶酶的激酶作用下转变 为纤维蛋白溶酶。 加之应纤溶酶的溶酶作用， 使血纤维蛋白发生 水解作用， 变成可溶性的小分子肽和氨基酸， 从而使加样处平板 形成半透明溶圈， 并根据圈的大小确定酶的活性单位。 在一定范 围内， 平板溶圈大小与浓度程线形关系， 因此测定纤维蛋白平板 的活性可看成纤溶酶和激活酶的双重作用。 该方法具有操作简 单， 溶解圈清晰、 易于观察， 重复性好等优点。 应用于本发明所涉及基因的基因工程纤溶酶蛋白的功能研 法、 气泡上升法、 精氨酸脂酶法。 在本发明的一个实施例中提供一种纤维蛋白平板法测定基 因工程纤溶酶的生物学活性的方法， 通过活性测定， 本发明的 基因工程菌株发酵液的活性单位超过 3,390,000 U/L。 在本发明的一个实施例中进一步提供了一种构建基因工程 菌株、 菌株为毕赤酵母 Pichia pastoris X-33 ( pPIC 6A-EFE ), CCTCC M204004。 在毕赤酵母菌中表达蚯蚓纤溶酶基因的方法， 该方法是用 pPIC 6A载体 ( Invitrogen公司）在毕赤酵母 X-33中 表达蚯蚓纤溶酶基因， 包括： （ 1 )蚯蚓纤溶酶基因的 PCR扩增； ( 2 ) PCR产物克隆至 T载体； （3 ) 穿梭表达质粒的构建； （4 ) 表达纤溶酶基因的酵母基因工程菌株的构建， 其步骤如下： ( 1 ) 蚯蚓纤溶酶基因的 PCR扩增： 以含有纤溶酶基因的 T 载体质粒为模板，根据 EFE成熟肽和表达载体 pPIC6A上的克隆 位 点 设 计 引 物 ， 上 游 引 物 P1 ： GAATTCGCATGGAACTTCCTCCCG (下划线处为内切酶 EcoR I 位点）， 下游引物 P2： TCTAGACGGTTTAGTTGTTGGTGAT (下 划线处为内切酶 Xba l位点）。 (3) Functional study and activity assay of genetically engineered plasmin protein There is currently no national standard for the determination of plasmin activity of humps, however, as a new drug, a method for measuring activity and potency is needed. At the same time, this method should be accurate, reliable and simple. Current methods of measurement can only be referenced to methods for determining the activity of other thrombolytic drugs. For example, 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 蚯蚓 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. In addition to the lysozyme action of plasmin, 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. Within a certain range, 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. In one embodiment 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 (Invitrogen), including: (1) 蚯蚓 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 蚯蚓 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 (underlined as the endonuclease Xba l site).
( 2 ) PCR产物克隆至 T载体：回收 PCR产物中的基因片段， 并克隆于 pGEM-T载体， 将该重组载体（ pGEM-T-EFE2 )转入大 肠杆菌 JM109中。 (2) 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.
( 3 ) 穿梭表达质粒的构建： 提取重组载体 pGEM-T-EFE2, 用 EcoR I 和 Xba l双酶切以得到目的片段 EFE;同样酶切 pPIC6A 空载体并在 CIAP作用下使之去磷酸化。 将去磷酸化后的线性化 的 pPIC6A空载体与欢酶切后得到的 EFE 在 T4DNA连接酶的作 用下 4Ό过夜， 以得到重组表达载体 pPIC6A-EFE, 将该重组载 体转入大肠杆菌 JM109中进行扩增。 (3) Construction of shuttle expression plasmid: The recombinant vector pGEM-T-EFE2 was extracted and digested with EcoR I and Xba l to obtain the target fragment EFE; the pPIC6A empty vector was also digested and dephosphorylated by CIAP. The dephosphorylated linearized pPIC6A empty vector and the EFE obtained by the enzyme digestion were subjected to T4 DNA ligase overnight to obtain a recombinant expression vector pPIC6A-EFE, and the recombinant vector was transferred into Escherichia coli JM109. Amplification.
( 4 )表达纤溶酶基因的酵母基因工程菌株的构建； a. 穿梭表达质粒的线形化： 提取重组盾粒 pPIC6A-EFE, 用 Sad酶切成线性， 同样酶切空质粒 pPIC6A作为对照。 b. 转化： 方法参考 Invitrogen公司表达载体说明书： 利用生 化方法将线性化的重组质粒 pPIC6A-EFE和空质粒 pPIC6A转化 X-33酵母菌株， 在含有杀稻瘟素（Blasticidin ) 300mg/ml的 YPD(4) Construction of yeast genetically engineered strain expressing plasmin gene; a. Linearization of shuttle expression plasmid: Recombinant Shield granule pPIC6A-EFE was extracted, cut into linear by Sad enzyme, and the empty plasmid pPIC6A was also digested as a control. b. Transformation: Method Reference Invitrogen Expression Vector Specification: The linearized recombinant plasmid pPIC6A-EFE and the empty plasmid pPIC6A were transformed into X-33 yeast strain by biochemical method, and YPD containing 300 mg/ml of blasticidin (Blasticidin)
( 1% 酵母抽提物、 2% 蛋白胨、 2%葡萄糖） 固体培养基上培养 2-3天， 直到有单菌落出现。 c. 筛选阳性转化子： 兆取 10-20个单菌落于 YPD液体培养 基中 30°C , 250rpm摇床培养直至对数生长晚期， 提取基因组进 行重组子的 PCR 定。 引物为 Invitrogen公司表达载体提供的通 用引物，其序列为上游： GACTGGTTCCAATTGACAAGC, 下游： GCAAATGGCATTC TGACATCC, 鉴定的阳性克隆命名为 Pichia pastoris X-33 ( pPIC 6A-EFE ), 在中国典型培养物保藏中心保藏 编号为 CCTCC M204004 , 在毕赤酵母中有两个编码乙醇氧化酶的基因 AOX1 和 AOX2 , 可以利用甲醇作为唯一碳源快速生长， 而当培养基中存 在其他碳源时， AOX基因不表达。 在毕赤酵母工程菌株中， 外 源基因被克隆至 AOX1启动子控制下， 因此必须以曱醇作为诱导 剂， 同时也是唯一碳源来实现外源蛋白的高效表达， 在诱导表达 时， 应补充甲醇以维持其 0.5%的含量。 在本发明的一个实施例中提供了一种毕赤酵母工程菌林诱 导表达的方法， 可用于毕赤酵母工程菌林诱导表达的培养基包括 但不仅限于下列培养基： MGY、 MM、 BMM、 BMMY, 各种培 养基的配置方法为本技术领域人员非常熟悉的方法， 可以通过参 考相关文献获得详情。 毕赤酵母工程菌株诱导表达方法如下： 挑取重组菌的单菌落 到 20mlYPD液体培养基， 30 °C、 250rpm培养进行活化； 16个小 时后取 10ml 活化后的菌液转接 500ml MGY液体培养基 30°C、 250rpm培养增浓；增浓 24小时后的菌液经离心收集菌体（ 1500g、 10分钟、 4 °C ); 将收集到的菌体重悬于 1000ml MM液体培养 基进行诱导表达， 每 24h添加甲醇至终浓度为 0.5%; 诱导 96h 后离心收集菌体（30000g、 20分钟、 4 °C ), 菌体采用玻璃珠破碎 法制备蛋白质抽提物用于活性测定。 当前尚无虹蚓纤溶酶活性测定的国家标准， 当前的测定方法 只能参考其它溶栓药物的活性测定方法。 比如尿激酶活性的气泡 上升法和测定蝮蛇抗栓酶的精氨酸脂酶法 （TAME ) 法， 但这两 种方法均不适用测定蚯蚓纤溶酶 , 因为纤溶酶具有纤维蛋白溶酶 原激活剂和蛋白水解酶的作用， 在溶解血栓时能发挥这两种功 能， 而且该酶的溶酶活性显著大于激酶活性。 纤维蛋白平板法能够客观地表示出纤溶酶溶栓的能力。 血纤 维蛋白平板中含有纤维蛋白溶酶原， 在纤溶酶的激酶作用下转变 为纤维蛋白溶酶。 加之应纤溶酶的溶酶作用， 使血纤维蛋白发生 水解作用， 变成可溶性的小分子肽和氨基酸， 从而使加样处平.板 形成半透明溶圈， 并才 据圈的大小确定酶的活性单位。 在一定范 围内， 平板溶圈大小与浓度程线形关系， 因此测定纤维蛋白平板 的活性可看成纤溶酶和激活酶的双重作用。 而运用基因工程的方 法则可以通过简单的发酵大量获得纤溶酶， 分离纯化工艺筒单， 产品纯度高， 成本底。 该方法具有操作简单， 溶解圈清晰、 易于 观察， 重复性好等优点。 在本发明的一个实施例中提供一种纤维蛋白平板法测定纤 溶酶的生物学活性的方法， 通过活性测定， 本发明的基因工程菌 株发酵液的活性单位超过 2, 900，000U/L。 本发明的一个实施例中提供了一种基因工程重组菌毕赤酵 母 X-33-pPIC6o;-EFE。 本发明进一步提供了基因工程纤溶酶蛋白在制备治疗心脑 血管疾病的药物中的应用。 本发明所涉及的基因工程蛋白在医疗、 卫生、 保健类药物的 研发领域中具有广泛的用途， 可用于制备治疗栓塞性心血管疾病 的冻干剂、 水针剂、 片剂、 栓剂、 喷雾剂等， 还可用于生产具有 预防血栓疾病、 有益心血管健康的保健品。 与传统的蚯蚓纤溶酶胶嚢制剂相比， 本发明所涉及的基因工 程药物有如下优点： （ 1 ) 蚯蚓纤溶酶胶嚢制剂口服给药作用緩 慢， 不能静脉注射， 不能应用于治疗急性心、 脑血管栓塞疾病； ( 2 )胶嚢制作工艺复杂， 需要人工大量养殖蚯蚓， 从虫体中分 离純化纤溶酶工艺烦瑣， 产品中可能含有各种小分子多肽或其它 化学物质， 可能引起机体的过敏反应。 而运用基因工程的方法则 可以通过简单的发酵大量获得纤溶酶， 分离纯化工艺筒单， 产品 纯度高， 成本底。 与其它溶栓类药物（包括生物提取的和基因工程药物）相比， 本发明所涉及基因工程药物有如下优点： (1% yeast extract, 2% peptone, 2% glucose) was incubated on solid medium for 2-3 days until a single colony appeared. c. Screening for positive transformants: 10-20 single colonies were taken from YPD liquid medium at 30 ° C, shaken at 250 rpm until late logarithmic growth, and the genome was extracted for PCR. 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. In the Pichia pastoris engineering strain, 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%. In one embodiment of the present invention, 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. The expression method of 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. At present, there is no national standard for the determination of rainbow plasmin activity, and the current measurement method can only refer to the activity determination method of other thrombolytic drugs. For example, 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 蚯蚓 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. In addition to the lysozyme action of plasmin, 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. Within a certain range, 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. In one embodiment of the present invention, there is provided 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. In one embodiment of the invention, 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. for treating embolic cardiovascular diseases. It can also be used to produce health care products with prevention of thrombotic diseases and beneficial cardiovascular health. Compared with the traditional sputum fibrinolytic enzyme preparation, 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. Compared with other thrombolytic drugs, including biologically extracted and genetically engineered drugs, the genetic engineering drugs of the present invention have the following advantages:
( 1 )链激酵 ( streptokinase SK ) 和尿激酶 ( urokinase UK ): SK和 UK属于第一代溶栓药物， 它们可以直接或者间接的激活 纤溶酶原使之转变成具有溶纤活性的纤溶酶。 SK是从 β型溶血 性链球菌培养液中提取的一种外源性纤溶酶原激活物， 是间接的 纤溶酶原激活剂。 UK是从人的尿液中分离出来的丝氨酸蛋白酶， 也可由人胚胎肾细胞培养液中获得。 这两种酶在实际使用过程存 在两个不可避免的问题： 首先， 他们作用的专一性差， 在溶解血 栓时常导致纤溶蛋白酶原的系统性激活， 而造成对凝固蛋白的无 差别性的消化， 增加了治疗过程中内出血的危险； 其次， 和底物 结合的亲和力小， 直接溶栓效果弱。 在治疗时使用剂量大， 导致 治疗成本昂贵。 SK 是一种非酶蛋白， 在使用中最大的问题是它 的抗原性。 几乎所有人的血液中或多或少都含有抗 SK抗体。 抗 体的存在一方面"中和"药物， 使药效降低， 另一方面可能引起过 敏反应。 (1) Streptokinase SK and urokinase UK: SK and 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 β-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. Increases the risk of internal bleeding during treatment; secondly, the affinity for binding to the substrate is small, and the direct thrombolytic effect is weak. Use a large dose during treatment, resulting in The cost of treatment is expensive. 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.
( 2 ) 組织型野溶醇原激活剂 （ tissue type plasminogen activator, tPA ): tPA是一种主要由血管内皮细胞产生的丝氨酸蛋 白酶，能催化无活性的纤溶酶原变成有活性的纤溶酶,使血栓溶 解。 正常人 tPA的血浆浓度约为 5ng/ml, 半衰期 5分钟。 tPA对 存在于血栓部分的纤溶酶原的特异性使用其做为第二代溶栓药 物受到重视， 在欧美发达国家， 用基因工程方法生产的 tPA已经 开始用于临床， 显示了一定的优越性。 但是由于半衰期短， 需要 反复大量用药才能奏效， 由此增加了系统性溶纤而导致内出血的 可能性， 且由于造价高而限制了它的广泛使用。 (2) tissue type plasminogen activator (tPA): 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. In developed countries in Europe and America, tPA produced by genetic engineering has begun to be used in clinical practice, showing a certain superiority. Sex. However, 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.
( 3 ) 其它的溶栓剂： 除了 UK, SK和 tPA之外， 目前研究 的还有蛇毒抗栓酶， 单链尿激酶型纤溶酶原激活物 （pro-UK ), 乙酰^ ^纤溶酶原链激酶激活性复合物（ APSAC )等。 但这些制剂 有的应用存在较大的副作用， 会发生过敏反应； 有的作用机理不 明， 使用万分复杂， 对制品品质要求极高， 有的价格昂贵。 生物提取的溶栓类药物在生产过程中， 需活性炭吸附， 主要 是用来脱色、 吸附热源、 除杂盾和助滤。 本发明所涉及的基因工 程蛋白純度高， 品质纯粹、 无热原， 避免脱炭过程和活性炭对环 境的污染， 生产成本低， 可直接用于生产注射用水剂、 粉剂。 本发明生产的基因工程纤溶酶可以与药学上可以接受的载 体 (如赋形剂、 填充剂、 吸收促进剂等）混合， 或者与其它药物混 合使用， 可以按照药学领域的常规方法将其制成所需的剂型， 可 通过口服、 注射等方式给药。 这些剂型包括但不仅限于冻干剂、 水针剂、 片剂、 栓剂、 喷雾剂。 本发明所涉及的基因工程药物可应用于心脑血管疾病的治 疗或辅助治疗中， 这些疾病包括但不仅限于： 缺血性脑病， 急性 心月几梗死合并高纤维蛋白原血症， 冠心病、 冠心病引起的高粘滞 血症、 冠心病心绞痛、 不稳定性心绞痛、 纤维蛋白原和血小板聚 集增高症， 本发明所涉及的基因工程药物还可应用于血液流变学 相关的一些疾病的治疗及辅助治疗中， 这些疾病包括但不仅限于 美尼尔病、 突发性耳聋、 肺心病、 糖尿病、 视网膜静脉阻塞、 肾 病综合症。 本发明所涉及的基因工程蛋白可以作为主要活性成分应用 于保健品的生产， 用于预防血栓疾病、 增进心脑血管健康。 上述以基因工程纤溶酶为主要活性成分的药物或其它生物 制品， 其制作过程或方法为该技术领域人员所熟悉， 生产过程应 依照国家相关的法律、 法规进行， 包括《中华人民共和国药典》(3) Other 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. However, some of these preparations have large side effects, and allergic reactions may occur; some of the mechanisms of action are unclear, the use is extremely complicated, and the quality of the products is extremely high, and some are expensive. Bio-extracted thrombolytic drugs require activated carbon adsorption during the production process, mainly for decolorization, adsorption of heat sources, removal of shields and filtration. Genetic worker involved in the present invention 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. 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
(国家药典委员会， 2000 ) 和 《中国生物制品规程》（中国生物 制品标准化委员会， 2000 )。 在本发明的实施例中， 提供了一种制备基因工程纤溶酶溶栓 药物制剂的方法， 并且在动物模型中进行了溶栓和对脑缺血保护 作用的试验， 结果表明本发明所涉及的基因工程纤溶酶有良好的 溶栓作用， 并对试险动物的脑缺血有保护作用， 这些都可以作为 例证说明本发明所涉及的基因工程纤溶酶在心脑血管疾病药物 研发领域中的用途。 全世界有心脑血管疾病人约 1500 万 ，所需的溶栓剂的潜在 市场约 2 0亿美元, 可用纤溶酶进行治疗的各类病人约 5000万 人， 潜在市场约 35亿美元， 因此， 基因工程纤溶酶具有广阔的 市场前景。 本发明提供一种基因工程菌株，毕赤酵母 Pichia pastoris X-33 ( pPIC6 a A-EFE ), 保藏单位： 中国典型培养物保藏中心， 保 藏日期： 2004年 1月 12 日， 保藏编号： CCTCC No: M204005。 本发明提供一种基因工程菌株，毕赤酵母 Pichia pastoris X-33 ( pPIC6 A-EFE ), 保藏单位： 中国典型培养物保藏中心， 保藏 曰期： 2004年 1月 12 曰， 保藏编号： CCTCC No: M204004。 实施例 下面结合附图及实施例对本发明作进一步说明， 倬并不作为 对本发明权利范围的限制。 所有实施例中的培养基及分子生物学操作方法为该领域技 术人员所熟悉，可以参考 Sambrook等 "分子克隆 "（实险室手册， 冷泉港， 1989 )及 "精编分子生物学实验指南" （美 /R奥斯伯等 著， 颜子颖等译， 北京， 科学出版社， 1998 ), 其全文并入本文 作为参考。 实施例 1. 蚯蚓纤溶酶的基因 PCR扩增及克隆 (National Pharmacopoeia Commission, 2000) and "Chinese Biological Products Regulations" (China Biological Products Standardization Committee, 2000). In an embodiment of the present invention, 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. These can be used as an example to illustrate the genetic engineering plasmin involved in the present invention in the field of cardio-cerebral vascular disease drug research and development. Use in. There are about 15 million people with cardiovascular and cerebrovascular diseases in the world, and the potential market for thrombolytics is about 2 billion US dollars. The number of patients who can be treated with plasmin is about 50 million, and the potential market is about 3.5 billion US dollars. , genetically engineered plasmin has broad market prospects. 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". (US/R Osbourne, et al., Yan Ziying, et al., Beijing, Science Press, 1998), which is incorporated herein by reference in its entirety. Example 1. PCR amplification and cloning of 蚯蚓 fibrinolytic enzyme gene
( 1 ) 扩增引物合成 才艮据 GeneBank 中已经发表的虫丘蚓纤溶酶 cDNA 序列设计 PCR 寡核苷酸扩增引 物。 上游引 物长 18 个核苷酸 ACATGGAACTTCCTCCCG， 下 游 引 物长 19 个核苷酸 ATCACCAACAACTAAACCG。 引物由上海生工生物工程有限公 司合成， 经 PAGE纯化。 ( 2 ) ：丘蚓总 RNA提取 取 3到 5条蚯蚓成体， 用 DEPC处理过的灭菌水漂洗后， 放 在铺有湿滤纸的平皿中饥饿过夜， 使其尽可能的吐尽体内的泥 沙。 再次用 DEPC水清洗干净后， 在液氮中研磨成粉末状， 按照 QIAGEN 公司 RNeasyR Mini Kit的产品说明书提取总 RNA。 将 提取到的 RNA溶液贮存于 -80Ό , 以备用。 (1) Amplification of primers 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. (2): Extracting 3 to 5 sputum from the total RNA of the scorpion, rinsing with DEPC-treated sterilized water, hunger overnight in a plate covered with wet filter paper, so that it can spit out the mud as much as possible. sand. After washing again with DEPC water, it was ground to a powder form in liquid nitrogen, and total RNA was extracted according to the product manual of QIAGEN RNeasyR Mini Kit. The extracted RNA solution was stored at -80 Torr for use.
( 3 )反转录合成 cDNA第一条链 按照 Promega 公司的 Reverse Transcription Reaction试剂盒 说明书进行， 以 Oligo(dT)20为引物合成 cDNA第一链。 具体操 作步骤如下： 将以下试剂加入一个经 DEPC浸泡并灭菌处理过的 PCR反应管中： 25mM MgCl2 12 μ 1; 緩冲液 6 μ 1; 10mM dNTP 6 μ 1; Recombinant RNasin Ribonuclease Inhibitor 2 μ 1; AVM反转 录酶 4 μ 1; Oligo(dT)20 6 μ 1； 丘蚓总 RNA 15 μ ΐ; 灭菌双蒸水 9 μ ΐο 反应条件为： 42°C 1小时； 95 °C 5分钟； 3 °C 5分钟。 (3) 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 μ 1; buffer 6 μ 1; 10 mM dNTP 6 μ 1; Recombinant RNasin Ribonuclease Inhibitor 2 μ 1; AVM reverse transcriptase 4 μ 1; Oligo (dT) 20 6 μ 1; 蚓 total RNA 15 μ ΐ; sterilized double distilled water 9 μ ΐο Reaction conditions: 42 ° C 1 hour; 95 ° C 5 Minutes; 3 °C for 5 minutes.
( 4 ) 目的基因的 PCR扩增 取 8μ∑ 反转录反应产物, 按照 Reverse Transcription Reaction试剂盒中给出的 PCR反应体系参数进行 PCR反应。 反应组分为：緩冲液 2.5 μ 1； 25mM MgCl2 1 .5 μ 1; l OmM dNTP 2μ1; 上游引物 Ι μΐ; 下游引物 Ιμΐ; mRNA反转录产物 15 μ 1; RT-PCR酶混合物 2 μ 1;灭菌双蒸水 5 μ 1。其反应参数为：(4) PCR amplification of the target gene Take 8 μ∑ reverse transcription reaction product, and carry out PCR reaction according to the PCR reaction system parameters given in the Reverse Transcription Reaction kit. The reaction components are: buffer 2.5 μ 1; 25 mM MgCl 2 1.5 μl; l OmM dNTP 2μ1; upstream primer Ι μΐ; downstream primer Ιμΐ; mRNA reverse transcription product 15 μ 1; RT-PCR enzyme mixture 2 μ 1; sterilized double distilled water 5 μ 1 . The reaction parameters are:
95°C预变性 4分钟； 94°C变性 1分钟， 55°C退火 1.5分钟， 72 °C延伸 2分钟， 20个循环； 94°C变性 1分钟， 55°C退火 1:5 分钟， 72°C延伸 2分钟， 10个循环； 72°C再延伸 10分钟。 1% 琼脂糖检测 PCR结果， 电泳结果如附图 1所示。 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, 20 cycles; denaturation at 94 °C for 1 minute, annealing at 55 °C for 1:5 minutes, 72 °C extended for 2 minutes, 10 cycles; 72 °C extended for another 10 minutes. 1% agarose detection PCR results, electrophoresis results are shown in Figure 1.
(5) 目的基因片段的回收 按照上海华舜生物工程公司 PCR产物回收试剂盒说明书回 收目的基因片段， 其过程如下： 取 30 1PCR 反应产物加入 1.4mlPB液（试剂盒提供：)， 混合物移入吸附柱中离心 15秒， 弃 废液； 在吸附柱中加入 400 μ 1PB液， 静置 1 分钟后， 离心 15 秒， 弃废液； 再加入 500μ1λ¥1液（试剂盒提供）， 离心 15秒， 弃废液，重复一次；在吸附柱中加入 300μ1Τ1液（试剂盒提供）， 静置 1分钟后， 离心 30秒， 收集离心液， 贮存于 -20°C。 (5) Recovery of the target gene fragment 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 μl of PB solution to the adsorption column, let stand for 1 minute, centrifuge for 15 seconds, discard the waste liquid; add 500μ1λ¥1 solution (provided by the kit), centrifuge for 15 seconds, discard The waste liquid was repeated once; 300 μl of Τ1 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.
(6) 目的基因片段克隆入载体 目的基因片段克隆入 pUCm-T载体，该载体购自上海生工生 物工程公司。 反应组分如下： PCR产物 5 μ 1; pUCm-T载体 1 μ 1; 10XT4DNA连接酶緩冲液 Ιμΐ; T4DNA连接酶 1 μ 1; 去离 子水 2μ1; 总体积为 10μ1， 4°C连接过夜。 感受态细胞的制备： 挑取单个 JM109菌落，接种于 3ml不含 氨苄青霉素的 LB培养基中， 37°C培养过夜， 次曰取上述菌液按 比例 1 : 100再接种于 50mlLB培养液中， 37°C振荡 3 小时， 待 菌液 OD值为 0.6时， 4°C 5000rpm离心 8分钟， 弃上清， 沉淀 用 0.1MCaCl2悬浮， 再 5000rpm离心 8分钟， 弃上清， 沉淀以适 量 0.1M CaCl2重悬， 分装后置水浴内 6小时备用。 连接产物的转化： 取上述连接产物 5 μ 1力口入 100 μ ΐ感受态 细胞水浴 60分钟， 42°C热休克 90秒， 再置冰浴 2分钟， 加入无 氨苄青霉素的 LB培养基 0.9ml, 37°C培养 1小时， 取 200 μ 1菌 液加入 40 μ 1 20mg/ml 5-溴 -4氯 -3吲哚（ D-半乳糖苷 （ X-gal ) ) 及 4 μ 1 0.1Μ异丙基硫代半乳糖苷 （IPTG ) 混勾后涂含氨苄青霉 素 LB平亚， 37°C培养 16小时，挑取白色菌落快速提取质粒进行 酶切鉴定， 酶切产物经 1%琼脂糖检测。 结果如图 2所示， 其阳 性克隆命名为 pUCm-T-EFE。 质粒 DNA的快速小量制备： 挑取单菌落接种于 4ml含氨苄 青霉素的 LB培养基， 37°C振荡培养过夜。 菌液加入 1.5ml离心 管中， 5000rpm离心 8分钟， 弃上清、 沉淀悬浮于 15 μ ΐ悬浮液 ( 50mM葡萄糖， 25mM Tris-HCl,10mMEDTA5PH8.0 ), 力口 200 μ 1裂解液（ 0.2MNaOH,l%SDS ), 置水浴 5分钟后， 加 150 μ ΐ中 和液（ 3.0MKAc,pH4.8 ), 冰浴 10分钟， 12000rpm离心 10分钟， 弃沉淀， 上清加等体积酚 /氯仿抽提一次， 上清加 2倍体积无水乙 醇， 12000rpm离心 10分钟， 沉淀物用 70%乙醇洗 1次， 真空抽 干加入 20 μ 1 TE ( lOmMTris- HC1， ImM EDTA, PH8.0和 1 μ ΐ RNaseA lOmg/ml ), -20 °C冻存备用。 (6) 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 μl; pUCm-T vector 1 μl; 10XT4 DNA ligase buffer Ιμΐ; T4 DNA ligase 1 μl; deionized water 2 μl; total volume 10 μl, 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 μ 1 into a 100 μ ΐ 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 μl of bacterial solution to 40 μl 20 mg/ml 5-bromo-4chloro-3-indole (D-galactoside (X-gal)) and 4 μl 0.1 Μ The propyl thiogalactoside (IPTG) was mixed with ampicillin LB, and cultured at 37 ° C for 16 hours. The white colonies were picked and quickly extracted and identified by enzyme digestion. The digested products were detected by 1% agarose. The results are shown in Figure 2, and the positive clone was named pUCm-T-EFE. Rapid miniprep preparation of plasmid DNA: Single colonies were picked and inoculated into 4 ml of ampicillin-containing LB medium and cultured overnight at 37 ° C with shaking. 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 μM suspension (50 mM glucose, 25 mM Tris-HCl, 10 mM EDTA 5 PH8.0), and a 200 μl lysate ( 0.2M NaOH, 1% SDS), after 5 minutes in a water bath, add 150 μM ΐ neutralizing solution (3.0 MKAc, pH 4.8), ice bath for 10 minutes, centrifuge at 12000 rpm for 10 minutes. Discard the precipitate, add the supernatant to an equal volume of phenol/chloroform, add 2 volumes of absolute ethanol to the supernatant, centrifuge at 12000 rpm for 10 minutes, wash the precipitate once with 70% ethanol, and vacuum dry to add 20 μl TE (10 mM Tris) - HC1, ImM EDTA, pH 8.0 and 1 μ ΐ RNaseA lOmg/ml ), stored frozen at -20 °C.
( 7 ) 丘蚓纤溶酶 cDNA序列测定 提取质粒 DNA双脱氧法测定核苷酸序列， 序列测定由上海 生工生物工程有限公司完成， 测序引物为 M13启动子引物。 测定结果： 丘蚓纤溶酶 cDNA序列长 747个核苷酸，腺嘌呤 核苷 （A )、 包嘧啶核苷 （C )、 鸟嘌呤核苷 （G ) 及胸腺嘧啶核 苷（Τ ) 含量分另 J为： A-185、 C-197、 G-189; T- 176 ( sequence No.l ), 编码 246个氨基酸（见 sequence Νο·2 )。 基因成熟肽序 列为第 3-740 位核苷酸， 其余核苷序列为非编码区。 该蛋白质 cDNA 成熟肽终止密码子位于基因第 741-743 位， 终止密码为 TAA。 根据蚯蚓溶纤酶 cDNA序列推测的该蛋白质等电点 （IP ) 为 4.15 , 蛋白质为酸' I"生。 (7) Hillside fibrinolytic enzyme cDNA sequencing The plasmid DNA was determined by the dideoxy method. The sequence was determined by Shanghai Shenggong Bioengineering Co., Ltd., and the sequencing primer was the M13 promoter primer. As a result, the cDNA sequence of the scorpion fibrinolytic enzyme is 747 nucleotides long, and the content of adenosine nucleoside (A), aglycone (C), guanosine (G) and thymidine (Τ) The other J is: A-185, C-197, G-189; T-176 (sequence No.l), encoding 246 amino acids (see sequence Νο·2). 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'.
( 8 ) 蚯蚓纤溶酶基因的修饰和改造 本发明所涉及的基因片段可以进行修饰， 比如突变基因的某 些核苷酸序列， 但并不影响其编码的蛋白质氨基酸序列； 可以增 加、 缺失本发明所涉及的氨基酸或者核苷酸序列， 这些修饰不会 影响蛋白质的生物化学特性、 高級结构和蛋白质的生物学功能， 比如： 可以在蛋白质或多肽 N-端或 C-端加上分泌信号肽， 还可 以加上适当的接头 （如 6个组氨酸）便于蛋白质提取、 纯化。 可 以人工合成全部或部分的核苷酸序列 , 合成的核苷酸序列序列可 以与本发明提供的序列有差异， 但其编码的蛋白质与本发明所涉 及的蛋白质序列相同， 这些可能的人工合成序列如下： atggarytnccnccnggnacnaarathgtnggnggnathgargcnmgnccntaygarttycc ntggc argtnwsngtnmgnmgnaarwsnwsngaywsnc ayttytgyggnggnwsnath athaaygaymgntgggtngtntgygcngcncaytgyatgcarggngargcnccngcnytn gtnwsnytngtngtnggngarcaygaymgnwsngcngcnwsnacngtnmgncaracnc aygaygtngaywsnathttygtncaygargaytayaayacnaayacnytngaraaygaygt nwsngtnathaaracnwsngtngcnathacnttygayathaaygtnggnccnathtgygcn ccngayccngcnaaygaytaygtntaymgnaarwsncartgywsnggntggggnacnat haaywsnggnggnathtgytgyccnaaygtnytnmgntaygtnacnytnaaygayacnac naaycartaytgygargaygtntayccnytnaaywsnathtaygaygayatgathtgygcnw sngayaayacnggnggnaaygaymgngaywsntgycarggngaywsnggnggnccny tnwsngtnaargayggnwsnggnathttywsnytnathggnathgtnwsntggggnathg gntgygcnwsnggntayccnggngtntaywsnmgngtnggnttycaygcngcntggath acngayathathacnaayaay 其中 m代表 a或 c; r代表 a 或 g ; w代表 a 或 t; s代表 c 或 g; y代表 c 或 t; k代表 g或 t; v代表 a 或 c 或 g; 代表 a 或 c 或 t; d代表 a 或 g 或 t ; b代表 c 或 g 或 t ; n代 表 g 或 a 或 t 或 c。 实施例 2.表达纤溶酶基因的毕赤酵母 Pichia pastoris X-33 (8) Modification and modification of the 蚯蚓plasmin gene 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. All or part of the 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 gtnwsnytngtngtnggngarcaygaymgnwsngcngcnwsnacngtnmgncaracnc aygaygtngaywsnathttygtncaygargaytayaayacnaayacnytngaraaygaygt nwsngtnathaaracnwsngtngcnathacnttygayathaaygtnggnccnathtgygcn ccngayccngcnaaygaytaygtntaymgnaarwsncartgywsnggntggggnacnat haaywsnggnggnathtgytgyccnaaygtnytnmgntaygtnacnytnaaygayacnac naaycartaytgygargaygtntayccnytnaaywsnathtaygaygayatgathtgygcnw sngayaayacnggnggnaaygaymgngaywsntgycarggngaywsnggnggnccny tnwsngtnaargayggnwsnggnathttywsnytnathggnathgtnwsntggggnathg gntgygcnwsnggntayccnggngtntaywsnmgngtnggnttycaygcngcntggath acngayathathacnaayaay Wherein m represents a or c; r represents a or g; w represents a or t; s represents c or g; y represents c or t; k represents g or t; v represents a or c or g; represents a or c or t; d represents a or g or t; b represents c or g or t; n represents g or a or t or c. Example 2. Pichia pastoris X-33 expressing the plasmin gene Pichia pastoris X-33
( pPIC6 a A-EFE )基因工程菌株的构建  Construction of (pPIC6 a A-EFE) genetically engineered strain
( 1 ) 蚯蚓纤溶酶基因的 PCR扩增 以 pUCm-T-EFE质粒为模板， 才艮据 EFE成熟肽和表达载体 p
Figure imgf000039_0001
(1) PCR amplification of 蚯蚓plasmin gene using pUCm-T-EFE plasmid as a template, based on EFE mature peptide and expression vector p
Figure imgf000039_0001
ATGGAACTTCCTCCCGGAACA (下划线处为内切酶 EcoR I 位点）。 下游引物 P2: GTCTAGATTAGTTGTTGGTGATGATGTC GGTGATCCATGCAGCAT (下划线处为内切酶 Xba I位点：)。 引 物由上海 Sangon生物工程公司合成， 合成产物经 PAGE纯化。 反应组分为： 緩冲液 2.5 μ ΐ; 25mM MgC12 1.5 μ ΐ; 10mM dNT P 2 μ 1; 上游引物 Ι μ ΐ; 下游引物 Ι μ ΐ; pUCm-T-EFE盾粒 1 μ 1; LA Taq DNA聚合酶 2 μ 1; 灭菌双蒸水 19 μ 1。 其反应参数为： 9 5 °C预变性 4分钟； 94°C变性 1分钟， 55 °C退火 1.5分钟， 72°C延 伸 2分钟， 30个循环； 72°C再延伸 10分钟。 (2) PCR产物克隆至 T载体 按照上海华舜生物工程公司 PCR 产物回收试剂盒说明书回 收目的基因片段，扩增产物克隆于 pGEM-T载体（Promega公司） 得到重組载体 GEM-T-EFE2，将该重组载体转入大肠杆菌 JM 109 中。 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 μ ΐ; 25 mM MgC12 1.5 μ ΐ; 10 mM dNT P 2 μ 1; upstream primer Ι μ ΐ; downstream primer Ι μ ΐ; pUCm-T-EFE Shield granule 1 μ 1; LA Taq DNA polymerase 2 μ 1; sterilized double distilled water 19 μl. 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. (2) The PCR product was cloned into the T vector and the target gene fragment was recovered according to the instructions of the PCR product recovery kit of Shanghai Huasheng Bioengineering Co., Ltd., and the amplified product was cloned into pGEM-T vector (Promega) to obtain the recombinant vector GEM-T-EFE2. This recombinant vector was transformed into E. coli JM109.
(3) 穿梭表达质粒的构建 提取重组载体 pGEM-T-EFE2, 用 EcoR I 和 Xba I双酶切以 得到目的片段 EFE; 同样酶切 pPIC6 Α空载体并在 CIAP作用 下使之去磷酸化。 将去磷酸化后的线性化的 pPIC6aA空载体与 双酶切后得到的 EFE 在 T4DNA连接酶的作用下 4°C过夜， 以得 到重组表达载体 PIC6 A-EFE, 将该重组载体转入大肠杆菌 JM109中进行扩增。 挑取白色菌落快速提取质粒进行酶切鉴定， 酶切产物经 1%琼脂糖检测结果如图 3所示。 (3) Construction of shuttle expression plasmid The recombinant vector pGEM-T-EFE2 was digested with EcoR I and Xba I to obtain the desired fragment EFE; the pPIC6 hollow vector was also digested and dephosphorylated by CIAP. The dephosphorylated linearized pPIC6aA empty vector and the double-cut EFE were subjected to T4 DNA ligase overnight at 4 ° C to obtain a recombinant expression vector PIC6 A-EFE, and the recombinant vector was transformed into Escherichia coli. Amplification was performed in JM109. The white colonies were picked and quickly extracted and identified by enzyme digestion. The results of the 1% agarose digestion of the digested products are shown in Fig. 3.
(4) 转化毕赤酵母 X-33 方法参考 Invitrogen 公司表达载体说明书 （ Pichia expression vectors for selection on blasticidin and purification of recombinant proteins manual )。 挑取毕赤酵母 X-33单菌落于接种 5毫升 YPD培养基， 30°C 250rpm摇床培养过夜，按 1%接种量接种 200mlYPD液体培养基， 30°C 250rpm摇床培养 3-5小时， 菌体浓度达到 0.8-1.0， 1500g离 心 10分钟， 沉淀用 10ml灭菌双蒸水重悬， 1500g离心 10分钟， 沉淀重悬于 0.4ml lOOmM的 LiCl中并转移至灭菌的 1.5ml离心 管中， 12000g离心 15秒， 弃上清， 加入 0.16ml 0.1M 的 LiCl重 悬， 备用。 提取重组盾粒 pPIC6 o A-EFE, 用 Sac I酶切成线性， 同样酶 切空质粒 pPIC6 c A作为对照。 取 50 μ 1上述感受态细胞， 12000g离心 15秒， 用移液器小 心吸取上清， 按顺序依次加入下列组分： 240 μ 1 50%PEG; 36 μ 1 1 M LiCl; 25 l 2 mg/ml 鲑鱼精 DNA; 50 μ 1 ( 5-10 g ) 线形 化重组质粒 pPIC6aA-EFE0以线形化空质粒 PIC6 α Α作为对照。 剧烈震荡一分钟使完全混勾， 30Ό静置 30分钟， 42Ό热激 20-25 分钟， 6000-8000rpm离心 15分钟， 沉淀用 lmlYPD培养基重悬， 30°C培养 1-4小时，取 25-100 μ ΐ图布于含有 300 μ g/ml杀稻瘟素 ( Blasticidin ) 的 YPD 固体培养基， 30°C培养 2-3 天， 直到有单 菌落出现。 ( 5 ) 筛选阳性转化子 挑取 10-20个单菌落于 YPD液体培养基中 30°C， 250rpm摇 床培养直至对数生长晚期， 提取基因組进行重組子的 PCR鉴定。 酵母基因組 DNA 制备方法： 取 1.5ml培养液 4 000 r/ 分钟 离.心 10 分钟。 菌体用液氮碾磨破壁。 加 7 mL DNA緩冲液（100 mmol/ L Tris HC1 , pH 8. 0， 10 mmol/ L EDTA ,1 % SDS) 混 匀， 65 °C保温 l h 。 lO OOO r/ 分钟离心 15 分钟。 上清用苯酚抽 提 2 次，每次 5 000 r/分钟 离心 7 分钟。再用氯仿抽提 1 次， 5 000 r/ 分钟离心 7 分钟。用 2. 5 倍乙醇沉淀 (力口 0. 3 mol/ L 醋酸钠 ,ρΗ 5. 2) ,10 000 r/ 分钟 离心 10 分钟。 70 %乙醇洗涤， 电吹风吹干， 加 TE 或水悬浮。 (4) 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 μl 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 μ 1 50% PEG; 36 μ 1 1 M LiCl; 25 l 2 mg/ Ml salmon sperm DNA; 50 μl (5-10 g) The linearized recombinant plasmid pPIC6aA-EFE 0 was linearized with the empty plasmid PIC6 α Α as a control. Vibrate vigorously for one minute to completely mix the hook, 30 Ό for 30 minutes, 42 Ό heat shock for 20-25 minutes, centrifuge at 6000-8000 rpm for 15 minutes, and the pellet was resuspended in 1 ml of YPD medium, and cultured at 30 ° C for 1-4 hours, taking 25- 100 μ ΐ 布 cloth was cultured in 300 μg/ml Blasticidin in YPD solid medium and incubated at 30 ° C for 2-3 days until a single colony appeared. (5) Screening positive transformants: 10-20 single colonies were picked and 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. 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, ρΗ 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.
PCR鉴定方法： 引物为 Invitrogen 公司表达载体提供的通 用引物，其序列为上游： G ACTGGTTCC AATTG AC AAGC； 下游： GCA AATGGCATTC TGACATCC。 PCR按常规方法进行， 扩增 条件为： 94 °C 45秒 ，54 °C 1 分钟 ，72 °C 1 分钟 ， 40个循 环。 扩增产物用用 0.8%琼脂糖检测其大小是否正确， 结果如附 图 3所示。 所 获 得 的 阳 性 克 隆 命 名 为 Pichia pastoris X-33(pPIC6aA-EFE) , 该菌株在中国典型培养物保藏中心保藏编 号为： CCTCC M204005。 实施例 3.基因工程纤溶酶蛋白的表达、 分离、 纯化 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
( 1 ) 毕赤酵母工程菌株 X-33(pPIC6aA-EFE)的发酵过程： a. L取重组菌的单菌落入 20mlYPD液体培养基（ 1% 酵母 抽提物、 2% 蛋白胨、 2%葡萄糖）， 30°C、 250rpm培养进行活化； b. 16个小时后取 10ml 活化后的菌液转接 500ml MGY液体 培养基液体培养基 30°C、 250rpm培养增浓； c. 增浓 24小时后的菌液经离心收集菌体（ 1500g、 10分钟、 4 ); d. 将收集到的菌体用重悬于 1000ml MM液体培养基进行诱 导表达， 每 24h添加曱醇至终浓度为 0.5%; e.诱导 96h后离心收集发酵液上清（ 30000g、 20分钟、 4 °C ); ( 2 )样品处理及 Ni2+柱层析过程： 发酵液上清灌装透析袋， 经以下步骤得柱层析样品； a. PEG20000 (聚乙二醇 20000 )掩埋透析袋浓缩， 浓缩约 10倍； b. 双蒸水洗净透析袋外表面， 将装有浓缩发酵液的透析袋 浸没在与原发酵液体积相等的 l x BINDING BUFFER ( 5mM imidazole+0.5M NaCl+20mM Tris · HCl PH=7.9 ) 内过夜， 以平衡 发酵液 PH、 离子浓度； c 同步骤 a, 浓缩至体积约为原发酵液的 1/10; d. 向透析袋中加入 l x BINDING BUFFER, 将浓缩过的样 品稀释 4倍； e. 同步骤 c; f. 从透析袋中取出浓缩发酵液， 并用少量 l xBINDING BUFFER将透析袋中的剩余样品洗净， 收集； g. 将浓缩发酵液高速离心（ 15000RPM, 15分钟）， 离心后 的上清用 0.45 u m微孔滤膜 （MILLIPORE公司） 过滤得到待柱 层析的样品。 注： 以上步 均在 4Ό进行。 (1) 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. The collected cells were resuspended in 1000 ml of MM liquid medium for induction, and sterol was added every 24 h to a final concentration of 0.5%; After 96 hours of induction, the supernatant of the fermentation broth was collected by centrifugation (30000g, 20 minutes, 4 °C); (2) Sample treatment and Ni2+ column chromatography process: The fermentation broth supernatant was filled with dialysis bags, and the column chromatography samples were obtained by the following steps. ; a. PEG20000 (polyethylene glycol 20000) buried in a dialysis bag concentrated, concentrated about 10 times; b. 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. Remove the concentrated fermentation broth from the dialysis bag and wash the remaining sample in the dialysis bag with a small amount of l xBINDING BUFFER g. The concentrated fermentation broth was centrifuged at high speed (15000 RPM, 15 minutes), and the supernatant after centrifugation was filtered through a 0.45 μm microporous membrane (MILLIPORE) to obtain a sample to be subjected to column chromatography. Note: The above steps are all performed at 4Ό.
Ni2+柱层析: a. Ni2+柱用 10床 1 x BINDING BUFFER, 10床无菌水清洗柱 床， 用 10床 l x CHARGE BUFFER ( 5Mm NiS04 ) 补 充 Ni2+, 再用 10床 l xBINDING BUFFER清洗柱床， 准备开始吸 附层析 （ 1床即是柱内介质的体积）； b.浓缩过的样品用蠕动泵以循环方式经过 Ni2+柱， 使带有 6 xHIS样品充分与 Ni2+结合， 过夜后从 Ni2+柱出口收集与 Ni2+ 反应后的样品 （穿漏液）； c.使 20床 1 X BINDING BUFFER流过 Ni2+柱 , 以冲洗留在 柱内的少量样品； d.用 5mM-100mM的咪唑溶液洗脱 Ni2+柱上非目的蛋白（洗 脱床数和洗脱溶液浓度视总蛋白含量和非目的蛋白与 Ni2+结合 能力灵活变化）注：如果按上面配方，则洗脱杂蛋白溶液为 60mM 咪唾。 e.用 1 x ELUTE BUFFER洗脱目的蛋白， 分步收集， 每 1床 (实际小于柱床体积）体积为一管， SDS-PAGE检睑目的蛋白在 洗脱液中 的分布。 Ni2+柱用 1 xELUTE BUFFER ( 1M imidazole+0.5M NaCl+20mM Tris■ HC1 PH=7.9 )继续洗脱， 洗掉 柱床中全部蛋白， 再用 1 X STRIP BUFFER ( 100Mm EDTA+0.5M NaCl+20mM Tris · HC1 PH=7.9 ) 洗去柱子上的 Ni2+离子。 ( 3 ) 牛内肠激酵降解 ( Enterokinase Cleavage ) 将 Ni2+柱纯化的纤溶酶用 1000 倍体积緩冲液 A ( 25mM Hepes/pH8.0, 5mM EDTA )透析 6-8小时后， 加入高度纯化的牛 内肠激酶 （ Sigma公司）， 进行降解反应， 反应条件如下： 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); c. a 20 bed 1 X BINDING BUFFER flowing through the Ni2+ column to flush a small amount of sample remaining in the column; d. eluting the non-target protein on the Ni2+ column with a 5 mM-100 mM imidazole solution ( 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. The Ni2+ column was eluted with 1 x ELUTE BUFFER (1M imidazole + 0.5M NaCl + 20 mM Tris ■ HC1 PH = 7.9), and all proteins in the bed were washed away, followed by 1 X STRIP BUFFER (100Mm EDTA + 0.5M NaCl + 20mM Tris) · HC1 PH=7.9) Wash off the Ni2+ ions on the column. (3) Enterokinase Cleavage Ni2+ column purified plasmin was dialyzed against 1000 volumes of buffer A (25 mM Hepes/pH 8.0, 5 mM EDTA) for 6-8 hours and then highly purified. The bovine enterokinase (Sigma) undergoes a degradation reaction with the following reaction conditions:
1份酶： 2000份底物 （重量比） 1 part enzyme: 2000 parts substrate (weight ratio)
37 °C反应 15-20小时后力 p入 P-Aminobenzawidine终止反应 Reaction at 37 °C After 15-20 hours, force p into P-Aminobenzawidine to stop the reaction
( 4 ) QAE-Toyopearl 550C阴离子交换树脂柱层析 柱子的制备： 将树脂混合均一后， 取出大约 50 亳升的树脂 加入緩冲液 A ( 25mM Hepes/pH8.0， 5mM EDTA ); 树脂沉淀后， 去掉上清液， 再加上等体积緩冲液 A, 于树脂混合均一后， 自然 沉淀， 这样重复 3 次。 固定好一支层析柱 ( 1.5 X 30cm ) 后， 堵 住柱出口 , 随后将緩冲液 A处理的树脂装入柱内。 然后打开柱子 下面出口后， 使树脂中緩冲液慢慢流出柱子， 然后用 200毫升緩 冲液 A, 在 4°C以下条件下， 继续平衡层析柱。 样品的制备： 将 10毫升 Ni2+亲和层析纯化的样品液， 装入 半透膜透析袋中， 用 2000毫升的緩冲液 A, 在 4°C以下条件， 进 行过夜透析。 纯化过程： （4°C以下温度操作）： 将透析后的样品加入层析 柱， 收集流出液； 用 3 X柱床体积 （ 150毫升） 的緩冲液 A洗柱 子； 用緩冲液 A和緩冲液 B (25mMHepes/pH8.0, 300mM aCl, 5mMEDTA), 通过一个线性梯度形成器， 制备的 NaCl浓度梯度 緩冲液洗脱层析柱。 （緩冲液 A 200亳升； 緩冲液 B 200毫升）； 洗脱样品用自动样品收集器以每部分 1 毫升体积分步收集。 用 OD280nm紫外光监测蛋白洗脱峰。 用 SDS-PAGE确认主蛋白峰 样品管含纤溶酶后， 将这些样混合。 (4) Preparation of QAE-Toyopearl 550C anion exchange resin column chromatography column: After mixing the resin uniformly, remove about 50 liters of resin into buffer A (25 mM Hepes/pH 8.0, 5 mM EDTA); after precipitation of the resin The supernatant was removed, and an equal volume of buffer A was added. After the resin was uniformly mixed, it was naturally precipitated, and this was repeated three times. After fixing one column (1.5 X 30 cm), block the column outlet, and then fill the resin treated with buffer A into the column. Then, after opening the outlet below the column, the buffer in the resin was slowly allowed to flow out of the column, and then the column was further equilibrated with 200 ml of buffer A under the conditions of 4 ° C or lower. Preparation of sample: A sample solution purified by 10 ml of Ni2+ affinity chromatography was placed in a semipermeable membrane dialysis bag, and dialysis was carried out overnight under conditions of 4 ° C with 2000 ml of buffer A. 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.
( 5 ) 純化产物的 SDS - PAGE电泳分析 a.凝胶的灌制：参照参见 Sambrook等 "分子克隆 "（实验室手 册， 冷泉港， 1989 ) 的方法， 配制 8%的分离胶溶液 15ml, 依次 混合各成分后加入催化剂 TEMED (N, N, Ν',Ν，四甲基乙二胺）， 立即混匀开灌胶， 封一层 0.1%SDS (二烷基^酸钠 )覆盖液面， 37°C下聚合 30分钟。 聚合完全后排净凝胶上的液体， 以同样方 法灌浓缩胶， 插入^ ^子， 避免产生气泡。 b.上样及电泳:在酵母诱导发酵液中加等体积 2 X SDS凝胶加 样缓冲液， 于 100°(^加热 3-5分钟变性， 按顺序加样， 最后在所 有不用的加样孔中加上等体积的加样緩冲液， 以 100V电泳至溴 酚兰进分离胶， 提高电压至 150V, 当溴酚兰到达底部前约 1cm 处结束电泳 （约需 4hr)。 c.凝胶染色:参见 《蛋白质純化与鉴定实验指南》（美国 D.R. 马歇克等著， 朱厚础等译， 科学出版社， 2002 )。 室温下， 凝胶 在旋转平台上緩慢摇动， 并进行下列步骤。 将凝胶在 50%的甲醛溶液中浸泡 2次， 每次 15分钟； 在 5% 的曱醛中浸泡 10分钟； 用双蒸水迅速洗 3次； 在 ΙΟ μ ηιοΙ/L二 石克苏糖醇 （DTT ) 溶液中浸泡 20分钟； 在 0.1%AgNO3 ( 0.1%, w/v ) 中进浸泡 20分钟； 先用双蒸水快速洗 1次， 然后用显影液 ( 15g碳酸 )溶于 500ml 7J 中， 用前力口入 250 μ 1 37% ( w/v ) 甲 醛溶液） 快速洗 2次， 每次不超过 15秒； 在显影液中浸泡数分 钟， 直至显出蛋白质带； 加入固体柠檬酸（每 200ml显影液加入 约 10g柠檬酸）， 停止显影； 用铝箔盖住凝胶继续浸泡 10分钟， 然后用双蒸水彻底清洗。 d.电泳结果:如附图 4所示。 实施例 4.基因工程纤溶酶蛋白的功能研究及活性测定 采用纤维蛋白平板法测定纤溶酶的生物学活性， 通过活性测 定， 发酵液的活性单位超过 3 E+06 U/L 。 图 5为测定时的纤溶 圈， 检测方法可参考郝苏丽等的方法 [郝苏丽， 沈佳， 蚓激酶生 物活性检测方法的研究， 中国药事 1996年第 10卷第 6期] , 具 体操作方法如下。 . ( 1 ) 铺板： 取纤维蛋白原液 （称取纤维蛋白原适量， 用 Tris— HC1 緩冲液配成每毫升中含 4.2mg 可凝蛋白的溶液） 12.7ml , 纤维蛋白溶酶原液（取纤维蛋白溶酶原用 Tris— HC1緩 冲液配成每毫升中含 lu的溶液） 0.67ml,琼脂液（称取琼脂 0. 8g 加 Tris— HC1緩冲液 100ml, 加热溶解） 13.4ml„ 凝血酶液（耳又 凝血酶用 Tris— HC1緩冲液配成含 70B. PU的溶液） 0.67ml。 混 匀倒入直径 9cm的有机玻璃盘内。 室温放置半小时。 凝好的板均 匀无气泡， 呈乳白色。 (5) SDS-PAGE electrophoresis analysis of the purified product a. Gelatinization: Refer to Sambrook et al., "Molecular Cloning" (Lab Handbook, Cold Spring Harbor, 1989) to prepare 15 ml of 8% separation gel solution, which are mixed sequentially. After the components, the catalyst TEMED (N, N, Ν', Ν, tetramethylethylenediamine) was added, and the glue was immediately mixed and sealed with a layer of 0.1% SDS (sodium dialkyl sulphate) to cover the liquid surface. Polymerization was carried out at ° C for 30 minutes. After the polymerization is completed, the liquid on the gel is drained, and the gel is immersed in the same manner, and inserted into the ^^ to avoid the generation of bubbles. b. Loading and electrophoresis: Add an equal volume of 2 X SDS gel loading buffer to the yeast-induced fermentation broth at 100° (^ heat for 3-5 minutes, add in the order, and finally in all unused samples) Add an equal volume of loading buffer to the well, electrophoresis at 100 V to the bromophenol blue into the separation gel, increase the voltage to 150 V, and end the electrophoresis (about 4 hr) about 1 cm before the bromophenol blue reaches the bottom. c. Gel staining: See the "Guidelines for Protein Purification and Identification Experiments" (US DR Marske et al., Zhu Houchu et al., Science Press, 2002). At room temperature, the gel was slowly shaken on a rotating platform and the following steps were performed. Soak the gel twice in 50% formaldehyde solution for 15 minutes each time; soak for 10 minutes in 5% furfural; wash 3 times quickly with double distilled water; in ΙΟ μ ηιοΙ/L distone sucrose Soak for 20 minutes in alcohol (DTT) solution; soak for 20 minutes in 0.1% AgNO 3 (0.1%, w/v); wash quickly with double distilled water, then dissolve in 500ml with developer (15g carbonic acid) In 7J, use a pre-forced 250 μl 37% (w/v) formaldehyde solution) to wash twice quickly, no more than 15 seconds each time; soak in the developer for a few minutes until the protein band is revealed; add solid lemon The acid (about 10 g of citric acid per 200 ml of developer solution) was stopped, the development was stopped; the gel was covered with aluminum foil for further 10 minutes, and then thoroughly washed with double distilled water. d. 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: . . (1) Laying: Take the fibrinogen solution (weigh the appropriate amount of fibrinogen, and mix it with Tris-HC1 buffer to form a solution containing 4.2 mg of coagulable protein per ml) 12.7 ml, plasminogen solution (take fibrin) Lysozyme was mixed with Tris-HC1 buffer to form a solution containing lu in ml) 0.67 ml, agar solution (weighed 0.8 g of Tris-HC1 buffer, dissolved in heat) 13.4 ml „ thrombin solution (The ear and thrombin were mixed with a solution containing 70B.PU in Tris-HC1 buffer) 0.67ml. Mix and pour into a plexiglass plate with a diameter of 9cm. Allow to stand at room temperature for half an hour. The plate is uniform and free of bubbles. milky.
( 2 )点样： 称取纤溶酶标准品适量。 用 Tris— HCI緩冲液配 成每毫升中含 2200 , 1100, 550, 270 四个浓度。 用啟量注射器 吸取 10 μ ΐ标准液点于板上。 点与点间应留有一定间距， 然后盖 上玻扳。 置 37 °C恒温培育 18小时， 去盖， 用游标卡尺测量其溶 圈的垂直直径， 以两直径乘积为从座标。 标准品单位为横座标， 在默对数座标纸上作图绘制标准曲线或求回归方程。供试品的单 位取在标准曲线范围内。 以供试品溶圈垂直两直径乘积， 在纤溶 酶标推曲线上查活力单位。 (2) Spotting: Weigh the appropriate amount of plasmin standard. Four concentrations of 2200, 1100, 550, and 270 per ml were used in Tris-HCI buffer. Use a starter syringe to draw 10 μL of standard solution onto the plate. There should be a certain distance between the points, and then cover the glass plate. Incubate at 37 °C for 18 hours, remove the cover, and measure the vertical diameter of the circle with a vernier caliper. The product of the two diameters is the ordinate. The standard unit is the abscissa, and the standard curve is drawn on the logarithmic coordinate paper or the regression equation is obtained. The unit of the test sample is taken within the range of the standard curve. The product is measured by the vertical diameter of the test strip, and the viability unit is checked on the fibrinolytic enzyme curve.
( 3 )检测结果： 两直径乘积为纵座标， 标准品单位为横座 标， 回归方程为 lgY = -1.573+0.544 IgX, Y为溶圈的垂直直径乘 积， 单位为 cm2, X为活性单位。 结果表明， 发酵液的活性单位 为 3.39 E+06 U/L。 实施例 5.表达纤溶酶基因的毕赤酵母 Pichia pastoris X-33 ( PIC6A-EFE )基因工程菌株的构建 (3) Test results: The product of two diameters is the ordinate, the standard unit is the abscissa, the regression equation is lgY = -1.573+0.544 IgX, Y is the product of the vertical diameter of the dissolved circle, the unit is cm 2 , and X is the active unit. . The results showed that the activity unit of the fermentation broth was 3.39 E+06 U/L. Example 5. Construction of Pichia pastoris X-33 (PIC6A-EFE) genetically engineered strain expressing plasmin gene
( 1 ) 蚯蚓纤溶酶基因的 PCR扩增 以 pUCm-T-EFE质粒为模板， 根据 EFE成熟肽和表达载体 pPIC6A 上的克隆位点设计引物， 上游引物 PI : GAATTC GCA TGG AAC TTC CTC CCG (下划线处为内切酶 EcoR I位点）， 下 游引物 P2： TCT AGA CGG TTT AGT TGT TGG TGA T (下划线 处为内切酶 Xba I位点）。引物由上海 Sangon生物工程公司合成， 合成产物经 PAGE纯化。反应组分为：緩沖液 2.5 μ 1; 25mM MgCl2 1.5 μ 1; 10mM dNTP 2 μ 1; 上游引物 1 μ 1; 下游引物 1 μ 1; pUCm-T-EFE质粒 1 μ 1; LA Taq DNA聚合酶 2 μ 1; 灭菌双蒸水 19 μ 1。 其反应参数为： 95 °C预变性 4min; 94°C变性 lmin， 55 °C 退火 1.5min， 72°C延伸 2min, 30个循环； 72 °C再延伸 10min。 (1) PCR amplification of 蚯蚓plasmin gene Using pUCm-T-EFE plasmid as template, 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 μ 1; 25 mM MgCl 2 1.5 μ 1; 10 mM dNTP 2 μ 1; upstream primer 1 μ 1; downstream primer 1 μ 1; pUCm-T-EFE plasmid 1 μ 1; LA Taq DNA polymerization Enzyme 2 μ 1; Sterilized double distilled water 19 μ 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.
( 2 ) PCR产物克隆至 T载体 按照上海华舜生物工程公司 PCR 产物回收试剂盒说明书回 收目的基因片段，扩增产物克隆于 pGEM-T载体（Promega公司） 得到重组载体 pGEM-T-EFE2,将该重组载体转入大肠杆菌 JM109 中。 ( 3 ) 穿梭表达质粒的构建 提取重组载体 pGEM-T-EFE2 , 用 EcoR I 和 Xba l双酶切以 得到目的片段 EFE; 同样酶切 pPIC6A空载体并在 CIAP作用下 使之去磷酸化。 将去磷酸化后的线性化的 pPIC6A空载体与双酶 切后得到的 EFE 在 T4DNA连接酶的作用下 4°C过夜， 以得到重 组表达载体 PIC6A-EFE, ^！夺该重组载体转入大肠杆菌 JM109中 进行扩增。 挑取白色菌落快速提取质粒进行酶切鉴定， 酶切产物 经 1%琼脂糖检测结果如图 3所示。 (2) The PCR product was cloned into the T vector and the target gene fragment was recovered according to the PCR product recovery kit of Shanghai Huasheng Bioengineering Co., Ltd., and the amplified product was cloned into pGEM-T vector (Promega) to obtain the recombinant vector pGEM-T-EFE2. This recombinant vector was transferred into E. coli JM109. (3) Construction of shuttle expression plasmid The recombinant vector pGEM-T-EFE2 was extracted and digested with EcoR I and Xba l to obtain the target fragment EFE; the pPIC6A empty vector was also digested and dephosphorylated under the action of CIAP. 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.
( 4 ) 转化毕赤酵母 X-33 方法参考 Invitrogen 公司表达载体说明书 （ Pichia expression vectors for selection on blasticidin and purification of recombinant proteins manual )。 挑取毕赤酵母 X-33单菌落于接种 5毫升 YPD培养基， 30°C 250rpm摇床培养过夜，按 1%接种量接种 200mlYPD液体培养基， 30°C 250rpm摇床培养 3-5小时， 菌体浓度达到 0.8-1.0, 1500g离 心 10分钟， 沉淀用 10ml灭菌双蒸水重悬， 1500g离心 10分钟， 沉淀重悬于 0.4ml lOOmM的 LiCl中并转移至灭菌的 1.5ml离心 管中， 12000g离心 15秒， 弃上清， 加入 0.16ml 0.1M 的 LiCl重 悬， 备用。 提耳又重组质粒 pPIC6A-EFE, 用 Sacl酶切成线性， 同样酶切 空廣粒 pPIC6A作为对照。 取 50 μ 1上述感受态细胞， 12000g离心 15秒， 用移液器小 心吸取上清， 按顺序依次加入下列组分： 240 μ 1 50%PEG; 36 μ 1 l M LiCl; 25 μ 1 2 mg/ml 鲑鱼精 DNA; 50 μ 1 ( 5-10 y g ) 线形 化重组质粒 pPIC6A-EFE。 以线形化空质粒 pPIC6A作为对照。 剧烈震荡一分钟使完全混匀， 30Ό静置 30分钟， 42°C热激 20-25 分钟， 6000-8000rpm离心 15分钟， 沉淀用 lmlYPD培养基重悬， 30°C培养 1-4小时，取 25-100 μ ΐ图布于含有 300 μ g/ml杀稻瘟素 ( Blasticidin ) 的 YPD 固体培养基， 30°C培养 2-3天， 直到有单 菌落出现。 利用生化方法将线性化的重组质粒 pPIC6A-EFE 和空质粒 pPIC6A转化 X-33酵母菌株 (参考 Picliia expression vectors for selection on blasticidin and purification of recombinant proteins manual, Invitrogen )。在含有 Blasticidin s Hcl( 300mg/ml )的 YPD 固体培养基上培养 2_3天， 直到有单菌落出现。 ( 5 ) 筛选阳性转化子 挑耳又 10-20个单菌落于 YPD液体培养基中 30°C , 250rpm摇 床培养直至对数生长晚期， 提取基因组进行重组子的 PCR鉴定。 所得到的阳' 1·生重组子均为 Mut+。 酵母基因组 DNA制备方法：取 1.5ml培养液 4 000 r/ min 离 心 10 min。菌体用液氮艰磨破壁。加 7 ml DNA緩冲液（100 m mol/ L Tris-HCl , pH 8. 0 , 10 m mol/ L EDTA ,1 % SDS) 。 混匀， 65 V 保温 1 h 。 10 000 r/ min离心 15 min。 上清用苯酚抽提 2 次,每 次 5 000 r/min 离心 7 min。 再用氯仿抽提 1 次， 5 000 r/ min离心 7 min。 用 2. 5 倍乙醇沉淀 (力口 0. 3 mol/ L 醋酸钠 ,ρΗ 5. 2) ,10 000 r/ min 离心 10 min。 70 %乙醇洗，电吹风吹干。 加 TE 或水悬浮， 力口 RNase。 (4) Transformation of Pichia pastoris X-33 The method is described in the Invitrogen expression vector 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. 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. Take 50 μl 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 μ 1 50% PEG; 36 μ 1 l M LiCl; 25 μ 1 2 mg /ml salmon sperm DNA; 50 μ 1 (5-10 yg) linearized recombinant plasmid pPIC6A-EFE. The empty plasmid pPIC6A was linearized as a control. Stir vigorously for one minute to completely mix, stand for 30 minutes at 30 °C, heat shock for 20-25 minutes at 42 °C, centrifuge for 15 minutes at 6000-8000 rpm, resuspend in 1 ml of YPD medium, and incubate at 30 ° C for 1-4 hours. 25-100 μ ΐ 布 cloth was cultured in 300 μg/ml Blasticidin in YPD solid medium and incubated at 30 ° C for 2-3 days until a single colony appeared. 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, ρΗ 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鉴定方法： 引物为 Invitrogen公司表达载体提供的通用 引物， 其序列为上游: GACTGG TTCCAATTGACAAGC; 下游： GCA AATGGCATTC TGACATCC。 PCR按常规方法进行， 扩增 条件为： 94 "C 45 s ,54 °C 1 min ,72 °C 1 min ,40个循环。 才广增 产物用用 0.8%琼脂糖检测其大小是否正确。 实施例 6.基因工程纤溶酶蛋白的诱导表达 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
( 1 ) 挑取重组菌的单菌落入 20mlYPD液体培养基 ( 1% 酵 母抽提物、 2% 蛋白胨、 2%葡萄糖）， 30°C、 250rpm培养进行活 化； (1) 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;
(2) 16个小时后取 10ml 活化后的菌液转接 500ml MGY液 体培养基（ 1.34% YNB、 4x10-5 %生物素、 1%甘油）30°C、 250rpm 培养增浓； (2) After 16 hours, 10 ml of activated bacterial solution was transferred to 500 ml of MGY liquid medium (1.34% YNB, 4x10-5 % biotin, 1% glycerol) at 30 ° C, 250 rpm to enhance concentration;
( 3)增浓 24小时后的菌液经离心收集菌体（ 1500g、 10min、(3) Enrichment After 24 hours, the bacterial liquid was collected by centrifugation (1500g, 10min,
4。C ); 4. C);
( 4 ) 将收集到的菌体用重悬于 1000ml MM 液体培养基 ( 1.34% YNB、 4x10-5 %生物素、 1%甲醇） 进行诱导表达， 每(4) The collected cells were resuspended in 1000 ml of MM liquid medium ( 1.34% YNB, 4x10-5 % biotin, 1% methanol) for induction.
24h添力口曱醇至终浓度为 0.5%; 24h to add sterol to a final concentration of 0.5%;
( 5 )诱导 96小时后离心收集菌体（ 30000g、 20min、 4 °C )0 (5) 96 hours after induction cells were harvested by centrifugation (30000g, 20min, 4 ° C ) 0
( 6) 在酵母消解酶緩冲液重悬菌体沉淀之前， 测定压紧细 包的体积， 以下所有步骤均在 4°C进行； ( 7 ) 用 1 体积的玻璃珠破菌緩冲液 （20mmol/L Tris-Cl, pH7.9, 10 m mol/L MgCl2, 1 m mol/L EDTA, 5%甘油， 1 m mol/L DTT, 0.3 m mol/L 酸按， 1 m mol/L PMSF ) 重悬细包； (6) 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)
(8) 用 2体积的玻璃珠破菌緩冲液与细胞混合， 加 4体积 冰冷的酸洗玻璃珠； (8) Mix 2 volumes of glass bead sterilizing buffer with cells, add 4 volumes of ice-cold pickled glass beads;
(9) 将细胞悬液转移至合适大小的带螺口的离心管中 （悬 液占离心管 0%-70%的容积），于 4°C以最大速度下振荡 30〜60s, 将管置于;水上放置 l~2min, 重复 3~5 次， 在显敖镜下检查破细 胞的程度， 继续步骤 10; (9) Transfer the cell suspension to a suitable size screw tube with a screw (the suspension accounts for 0%-70% of the volume of the centrifuge tube), and shake at a maximum speed for 30~60s at 4°C. Place on the water for 1~2min, repeat 3~5 times, check the degree of cell destruction under the fluoroscopy, continue to step 10;
( 10)沉淀玻璃珠， 轻轻倒出上清， 加入 2〜4体积的玻璃珠 破菌緩冲液， 颠倒管 5~10次， 等玻璃珠沉淀后轻轻倒出上清， 合并上清液； (10) Precipitate the glass beads, gently pour out the supernatant, add 2~4 volumes of glass bead sterilizing buffer, invert the tube 5~10 times, wait for the glass beads to precipitate, then pour out the supernatant, and combine the supernatant Liquid
( 11 )合并的上清液于 4°C, 12000g 离心 60min, 收集上清 即为抽提液。 长期贮存时， 将其分成小份于液氮中快速冷冻， - 80°C贮存。 实施例 7. 基因工程纤溶酶蛋白的活性测定 采用纤维蛋白平板法测定基因工程纤溶酶的生物学活性， 通 过活性测定， 发酵液的活性单位超过 2.9E+06U/L。 图 6为测定 时的溶圈， 检测方法可参考郝苏丽等的方法 [郝苏丽， 沈佳， 蚓 激酶生物活性检测方法的研究，中国药事 1996年第 10卷第 6期] , 其全文参考并入本文。 具体操作过程如下。 (11) The combined supernatant was centrifuged at 12000 g for 60 min at 4 ° C, and the supernatant was collected as an extract. For long-term storage, it is divided into small portions and rapidly frozen in liquid nitrogen, and stored at -80 °C. 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. [Hao Suli, Shen Jia, Research on the Detection Method of Bioactivity of Prion Kinase, Chinese Pharmacy, Vol. 10, No. 6, 1996], the full text of which is incorporated herein by reference. The specific operation process is as follows.
( 1 ) 铺板： 取纤维蛋白原液 （称取纤维蛋白原适量. 用 Tris— HC1 緩冲液配成每毫升中含 4.2mg 可凝蛋白的溶液） 12.7ml , 纤维蛋白溶酶原液（取纤维蛋白溶酶原用 Tris— HC1緩 冲液配成每毫升中含 lu的溶液） 0.67ml, 琼脂液（称取琼脂 0.8g 加 Tris— HC1緩冲液 100ml, 力 P热溶解， 滤过） 13.4ml. 凝血酶 液（取凝血酶用 Tris— HC1緩冲液配成含 70B. PU的溶液）0.67ml。 混匀， 倒入直径 9cm的有机玻璃盘内， 室温放置半小时。 凝好的 板均勾无气泡， 呈乳白色。 (1) Plating: Take the fibrinogen solution (weigh the appropriate amount of fibrinogen. Prepare a solution containing 4.2 mg of coagulable protein per ml with Tris-HC1 buffer) 12.7ml, plasminogen solution (take fibrin) Lysozyme was mixed with Tris-HC1 buffer to form a solution containing lu in ml) 0.67ml, agar solution (weigh 0.8g of agar and 100ml of Tris-HC1 buffer, heat P is dissolved, filtered) 13.4ml Thrombin solution (take thrombin into Tris-HCl buffer to prepare a solution containing 70B. PU) 0.67 ml. Mix well, pour into a plexiglass plate with a diameter of 9 cm and leave it at room temperature for half an hour. The condensed plates are free of air bubbles and are milky white.
( 2 ) 点样： 称取纤溶酶标准品适量。 用 Trios— HCI緩冲液 配成每亳升中含 2200, 110 , 550, 270 四个浓度。 用^:量注射器 吸取 10 μ ΐ标准液点于板上。 点与点间应留有一定间距， 然后盖 上玻扳. 置 37°C恒温培育 18小时， 去盖， 用游标卡尺测量其溶 圈的垂直直径， 以两直径乘积为纵座标. 标准品单位为横座标， 在又对数座标纸上作图绘制标准曲线或求回归方程。供试品的浓 度取在标准曲线范围内， 以供试品溶圈垂直两直径乘积在纤溶酶 标推曲线上查活力单位。 ( 3 ) 检测结果： 两直径乘积为纵座标， 标准品单位为横座 标，回归方程为 lgY = -1.334+0.479 lgX， Y为溶圈的垂直直径积， 单位为 cm2, X为活性单位。 结果表明， 发酵液的活性单位为 2.9 E+06 U/L。 实施例 8基因工程重組菌毕赤酵母 X-33-pPIC6o;-EFE 培养基基本配方： (2) Spotting: Weigh the appropriate amount of plasmin standard. The concentration of 2200, 110, 550, 270 in each liter was determined by using Trios-HCI buffer. Pipette 10 μ ΐ standard solution onto the plate with a ^: syringe. A certain distance should be left between the points, and then covered with a glass plate. Incubate at 37 °C for 18 hours, remove the cover, measure the vertical diameter of the solution circle with a vernier caliper, and use the product of the two diameters as the ordinate. Standard unit For the abscissa, plot a standard curve or regression equation on a logarithmic coordinate paper. The concentration of the test sample is taken within the range of the standard curve, and the vertical diameter of the test solution is checked by the two diameter products on the plasmin-enhanced curve. (3) Test results: The product of two diameters is the ordinate, the standard unit is the abscissa, the regression equation is lgY = -1.334+0.479 lgX, Y is the vertical diameter product of the dissolved circle, the unit is cm 2 , X is the active unit . The results showed that the activity unit of the fermentation broth was 2.9 E+06 U/L. Example 8 Genetically engineered recombinant Pichia pastoris X-33-pPIC6o;-EFE medium basic formula:
Figure imgf000057_0001
培养基用 30%氨水调 pH至 6.0, 121 °C在位灭菌 20 min,灭菌 完成后加入过滤除菌的 PTMi至终浓度为 4.35/L。 PTM2盐溶液:
Figure imgf000057_0001
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:
Figure imgf000058_0001
Figure imgf000058_0001
ΡΤΜ 溶液经 0.22μηι :孑 ϋ滤膜过滤除菌。 接种量： 10%MGY种子 发酵阶段控制： 发酵罐诱导毕赤酵母表达基因工程蛋白 EFE 过程分为工程菌的扩培和目的蛋白的诱导表达两个阶段，。 从新鲜划线接种的平板上挑一个单菌落于 500 MGY培养基 中 30°C, 300rpm摇床培养约 36h 至 OD6QO 为 15 作为种子菌。 将该种子菌接种于 5升已除菌的基本盐培养基中进行菌浓扩培。 在工程菌的扩培阶段温度控制在 29 °C ,调节通气和转速使溶氧不 低于 25%。 菌浓扩培进行到约 23.5 h后， 溶氧快速上升表明培养 基中的甘油已耗尽。 开始以 75mL/h 的速度补充甘油 （50%w/v, 12 mL/LPTMl )， 该阶段持续约为 6 h至湿菌重达到 200 g/L。 终止甘油的补力口， 一次性加入曱醇至终浓度 0.5%, 观察溶氧 水平变化， 当溶氧开始上升时开始用蠕动泵自动补加甲醇， 才艮据 保持溶氧水平不低于 25%的原则调节甲醇流加速率， 最初 4 h甲 醇 （ 100% , 12mL/L PTMl ) 的补加速度为 15mL /h, 随后的两小 时为 40 mL/h, 最后补加速度被控制在 50mL/h, 保持该速度至 诱导表达约 83.5 h后发酵结束， 此时测得湿菌重为 270g/L, 收 集发酵液， 4000rpm离心 15min收集上清， -20 °C保存备用。 在诱导过程中， 每 6小时取样检测单位体积内的湿菌重及表 达产物的活性。 诱导 48小时后湿菌重已经达到 230 g/L， 并能够 检测到表达产物的活性， 表明工程蛋白已开始表达。 发酵液经超滤， （NH4 ) 2S04盐析后都有纤溶活性。 以盐析 样品估算， 与标准天然 EFE比较， 16小时纤维蛋白平板上每升 约含有 150万单位以上的纤溶活性。 实施例 9. 基因工程纤溶酶制备溶栓类药物和 ΡΤΜ The solution was sterilized by filtration through a 0.22 μηι : 孑ϋ 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. In the expansion stage of the engineering bacteria, 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%. After the bacteria were concentrated and cultured for about 23.5 h, 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. Stop the filling port of glycerin, add sterol to the final concentration of 0.5% at a time, observe the change of dissolved oxygen level, start to use the peristaltic pump to automatically add methanol when the dissolved oxygen starts to rise, and then keep the dissolved oxygen level below 25 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. During the induction process, 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 . Estimated by salting out the sample, compared to the standard natural EFE, 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
制备口服类溶栓药物 将纯化的纤溶酶（方法见实施例 2， 实施例 5 , 实施例 8 )在 緩冲液（23克 甘氨酸 /升， 1.6克磷酸氢二钠 /升， 0.55克磷酸二 氢钠 /升， pH7.0 ) 透析后， 用超滤浓缩到每毫升含 1亳克浓度， 用 0.22 μ πι孔径的膜过滤灭菌。 然后， 大约 1 亳升样品液（约 300000 活力单位） 装瓶， 冷冻干燥， 制成成品。 使用时， 样品 首选用 1亳升医用水溶解， 用作生物测活和注射病人。 基因工程 纤溶酶样品规格为： 300000 U 基因工程纤溶酶； 23mg甘氨酸； 1.6mg 磷酸氢二钠； 0.55mg磷酸二氢钠。 另外配备 5 毫升医用 级水。 将基因工程发酵产物破碎（方法见实施例 5 ), 然后收集上清, 在緩冲液（23克 甘氨酸 /升， 1.6克磷酸氢二钠 /升， 0.55克磷酸 二氢钠 /升， pH7.0 ) 中透析， 最后用超滤浓缩到每毫升含 1毫克 浓度， 用 0.22 μ ιη孔径的膜过滤灭菌。 滤液可直接最为口服水剂 供病人服用， 或者大约 1毫升滤液（约 300000 活力单位）装瓶， 冷冻干燥， 制成粉剂成品， 使用时温水溶解口月艮。 实施例 10. 基因工程纤溶酶蛋白制剂的应用 鼠抗凝实验 方法参考陈健康等的文献进行 [陈健康， 王雷， 李珂， 郭峰， 杨军， 艮杏黄酮与基因工程纤溶酶的抗凝溶栓作用， 心脏杂志 2001,13(4): 308-309] , 其全文参考并入本文。 挑选昆明种雄性小鼠 30只， 随机分为 3组， 体重 20土 2g。 连续给药 3天， 各组分别注射 5000U/kg基因工程纤溶酶， 0.2mg 阿司匹林,对照组注射生理盐水 0.5mL。第 3天用药后 2小时开始 实验。 将小鼠尾尖部剪断 lcm， 间隔 10s用滤纸吸去血清,不得挤 压,记录小鼠出血时间。 实验结果 （见表 1 )表明， 基因工程纤溶酶组和阿司匹林组 出血时间均延长， 基因工程纤溶酶用药组和对照组比较有显著性 意义,说明基因工程纤溶酶有艮好的抗凝血作用。 实验小鼠尾部出血时间对照表 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 μm. 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 μm 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. After continuous administration for 3 days, 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
Figure imgf000061_0001
Figure imgf000061_0001
(与^"照^ 目比， p <0.05 ) 实施例 11. 基因工程纤溶酶蛋白制剂的应用——对大鼠实险性 脑缺血的保护作用 (Compared with ^"Photos, p <0.05) Example 11. Application of genetically engineered plasmin protein preparation - protective effect on cerebral ischemia in rats
( 1 ) 大鼠大脑中动脉闭塞模型 （MCAO ) 的制备 方法参照文献进行 [Tamura A, Graham DI, McCulloch J, Teasdale GM.， Focal cerebral ischaemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion.J Cereb Blood Flow Metab. 1981;1(1):53-60.] [冯亦璞，闰韶华， 小鼠短暂性 MCAO模型， 张均 田， 现代药理实验方法， 北京医科大学中国协和医科大学联合出 版社， 1998]。 用 10%水合氯醛 (300mg/kg)ip麻醉大鼠， 仰位固定， 颈部正 中切口，暴露右侧颈总动^ 由分叉处向头端依次游离， 结扎并剪 断枕骨下动脉和甲状腺上动脉， 在颈外动脉远端结扎并切断， 游 离备用。 分离颈内动脉， 用丝线在颈外动脉根部打一松扣， 夹闭 颈总动月永和颈内动月永， 用细线经颈外动^主干切口， 緩' [·曼向颈内 动脉入颅方向推进， 以颈总动脉分叉处为标记， 推进 20 亳米左 右。 扎紧颈外动脉根部松扣。 一小时后,小心拔出细线， 扎紧动脉 残端， 用外科无损伤缝合线缝合皮肤， 完成大脑中动脉栓塞。 术 中术后室温控制在 22 ± 1 °C。 雄性 Wister大鼠（50 ± 50 g )，随机分成 4个组，即 殳手术组、 模型组、 基因工程纤溶酶 （ 2500U/kg ) 组， 基因工程纤溶酶 ( 5000U/kg )组。其中假手术组暴露左大脑中动脉和左颈总动脉， 不结扎。 各给药组于结扎前 10分钟分别静脉注射基因工程纤溶 酶， 模型组则给予生理盐水 0.5ml。 (1) Preparation method of rat middle cerebral artery occlusion model (MCAO) is carried out according to the literature [Tamura A, Graham DI, McCulloch J, Teasdale GM., Focal cerebral ischaemia in the rat: following: _ _ _ _ _ _ _ _ _ Middle cerebral artery occlusion.J Cereb Blood Flow Metab. 1981;1(1):53-60.] [Feng Yizhen, Yan Hua, Mouse Transient MCAO Model, Zhang Juntian, Modern Pharmacological Experimental Method, Beijing Medical University, China Union Medical University, United Press, 1998]. 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. [··Man to the internal carotid artery Advance in the direction of the cranial, marked with the bifurcation of the common carotid artery, and advance about 20 亳. Tightening the root of the external carotid artery. One hour later, the thin wire was carefully pulled out, the arterial stump was tightened, and the skin was sutured with a surgically intact suture to complete the middle cerebral artery embolization. The room temperature was controlled at 22 ± 1 °C after operation. Male Wister rats (50 ± 50 g) were randomly divided into 4 groups: the sputum operation group, the model group, the genetically engineered plasmin (2500 U/kg) group, and the genetically engineered plasmin (5000 U/kg) group. 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.
( 2 )基因工程纤溶酶对脑缺血大鼠存活时间的影响 实验结果见表 2 表 2 基因工程纤溶酶对脑缺血大鼠存活时间的影响 (2) The effect of genetic engineering plasmin on the survival time of cerebral ischemia rats is shown in Table 2. Table 2 Effect of genetic engineering plasmin on survival time of cerebral ischemia rats
Table 2 Effect of Lumbroinase on survival time in ischemic mice ( ,x 土 S ) 分 組 例数 (n) 存活时间 （min) 对照组 15 9.5 ±4.1 支手术组 15 8.2 + 5.4 基因工程纤溶酶 ( 2500U/kg ) 15 11 ±8 基因工程纤溶酶 ( 5000U/kg ) 15 68±282 与^"照^ ^目比， p < 0.001 Table 2 Effect of Lumbroinase on survival time in ischemic mice ( , x soil S ) group number (n) survival time (min) control group 15 9.5 ±4.1 surgery group 15 8.2 + 5.4 genetic engineering plasmin (2500U/kg 15 11 ±8 genetically engineered plasmin (5000U/kg) 15 68±28 2 and ^"photo^^, p < 0.001
(3)基因工程纤溶酶对脑缺血大鼠大脑动脉 24h后梗死面 积和脑组织含水量的影响， 实验结果见表 3 (3) The effect of genetically engineered plasmin on the infarct size and brain water content of cerebral arteries in rats with cerebral ischemia for 24 hours. The experimental results are shown in Table 3.
表 3 基因工程纤溶酶对小鼠大脑中动脉闭塞 24h后梗死面积及 组织的含水量 Table 3 Infarct size and tissue water content after 24 h of cerebral artery occlusion in mice by genetically engineered plasmin
Inhibitory effect of lumbrokinase on infarction area and water content in brain ofter 24 h MCAO in rats ( x 士 S ) 分 组 例数 (n) 梗死面积（ 1/2) 含水量 （mL/g) 手术组 8 1.22 ±0.05 3.74 ±0.09 模型组 8 7.17±0.37e 4.99士 0.45c 基因工程纤溶酶 Inhibitory effect of lumbrokinase on infarction area and water content in brain ofter 24 h MCAO in rats (x S) group number (n) infarct size (1/2) water content (mL/g) surgery group 8 1.22 ±0.05 3.74 ±0.09 model group 8 7.17±0.37 e 4.99 0.45 c genetic engineering plasmin
8 5.77±0.22bd 4.77 ±0.348 5.77±0.22 bd 4.77 ±0.34
( 2500U/kg ) 基因工程纤溶酶 (2500U/kg) genetically engineered plasmin
8 4.07±0,65b 4.41 ±0.56a ( 5000U/kg ) 8 4.07±0,65 b 4.41 ±0.56 a ( 5000U/kg )
与模型组比较， a: p <0.05, b: ρ < 0.01; 与假手术组比较， c: p < 0.001; 与模型 +基因工程纤溶酶 2500U/kg和 5000U/kg组 比较， d: p <0.05。 Compared with the model group, a: p <0.05, b: ρ < 0.01; compared with the sham operation group, c: p < 0.001; compared with the model + genetic engineering plasmin 2500 U/kg and 5000 U/kg, d: p <0.05.
(4)基因工程纤溶酶对大鼠大脑中动脉闭塞 24小时后神经 功能障碍的影响 上述 3组动物在大脑中动脉结扎术后 24小时观察其神经功 能障碍的情况， 将神经功能障碍分为 5级： I级为无神经系统功 能损害， 记 1分； II级仅有轻度功能障碍， 表现为行走时虽不用 扶持， 但步态欠稳， 提尾时脑损伤对侧前肢屈曲， 记 2分； III级 有中度功能障碍， 表现为在扶持下方可站立， 不持久， 提尾时脑 损伤对侧前肢屈曲， 向手术侧转圈， 记 3分； IV级为重度功能障 碍， 表现为有意识变化， 嗜睡， 反应迟飩， 不能站立， i己 4分； V级为死亡， i己 5分。 该方法为溶栓类药物常用的测定方法， 可 参考 [Lawner PM, Laurent JP, Simeone FA, Fink EA ,Effect of extracranial-intracranial bypass and pentobarbital on acute stroke in dogs. J Neurosurg. 1982 Jan;56(l):92-96.][Diaz FG, Mastri AR, Ausman JI, Chou SN., J Neurosurg. Acute cerebral revascularization after regional cerebral ischemia in the dog., 1979 Nov;51(5):644-653.] , 其全文参考并入本文。 实验结果见表 4。 (4) Effects of genetically engineered plasmin on neurological dysfunction after occlusion of middle cerebral artery in rats for 24 hours. The above three groups of animals were observed for neurological dysfunction 24 hours after ligation of middle cerebral artery, and the neurological dysfunction was divided into two groups. 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. This method is commonly used for the determination of thrombolytic drugs, and can be referred to [Lawner PM, Laurent JP, Simeone FA, Fink EA, Effect of extracranial-intracranial bypass and pentobarbital on acute stroke in dogs. J Neurosurg. 1982 Jan; 56 (l ): 92-96.] [Diaz FG, Mastri AR, Ausman JI, Chou SN., J Neurosurg. Acute cerebral revascularization after regional cerebral ischemia in the dog., 1979 Nov; 51(5): 644-653.] , The full text is incorporated herein by reference. The experimental results are shown in Table 4.
基因工程纤溶酶对小鼠大脑中动脉闭塞 24h后 神经功能障碍的影响 Effect of genetically engineered plasmin on neurological dysfunction after 24 h occlusion of middle cerebral artery in mice
Table 4 Influence of lumbrokinase on neurologic deficits ( ND ) scores produced by 24h MCAO in rats ( x 土 S ) 分 组 例数 (n) 神经功能障碍评分 模型组 8 4.21 ±0.3d 假手术组 8 1.45 + 0.5 基因工程纤溶酶 Table 4 Influence of lumbrokinase on neurologic deficits ( ND ) scores produced by 24h MCAO in rats ( x soil S ) grouping cases (n) neurological dysfunction scoring model group 8 4.21 ±0.3 d sham group 8 1.45 + 0.5 genetic engineering fiber Lysozyme
8 2.16±0.5b 8 2.16±0.5 b
( 2500U/kg ) 基因工程纤溶酶  (2500U/kg) genetically engineered plasmin
8 1.67±0.3b 8 1.67±0.3 b
( 5000U/kg )  (5000U/kg)
与模型组比较， b: p <0.01； 与 支手术组比较， d: p <.001。 Compared with the model group, b: p <0.01; compared with the surgery group, d: p <.001.
(5)基因工程纤溶酶对小鼠大脑中动脉闭塞 24小时后脑组 织总抗氧化能力、 NO含量及 NOS活性的影响 实验结果见表 5。 表 5 基因工程纤溶酶对小鼠大脑中动脉闭塞 24h后脑组织总抗 氧化能力， 一氧化氮含量和一氧化氮合酶活性的影响 (5) The effect of genetic engineering plasmin on the total antioxidant capacity, NO content and NOS activity of brain tissue after 24 hours of occlusion of middle cerebral artery in mice. Table 5 Effects of genetically engineered plasmin on total antioxidant capacity, nitric oxide content and nitric oxide synthase activity in brain tissue of mice after occlusion of middle cerebral artery for 24 hours
Table Effect of Lumbrokinase on T-AOC. NO content and NOS activity after 24h MCAO ( n=6, x 士 S ) ) 总抗氧化能力 NO NOS 分 组 Table Content of Lumbrokinase on T-AOC. NO content and NOS activity after 24h MCAO (n=6, x S S ) ) Total antioxidant capacity NO NOS group
( kU/g ) ( μ mol/g) (kU/g) 正常对照 3.17 ±0.56 1.11 ±0.20 039 ±0.06 模型组 1.20±0.15d 3.20±0.54d 0.84±0.79d 假手术组 3·16±0.58 1.09 + 0.20 0.41 ±0.04 基因工程纤溶酶 (kU/g) (μ mol/g) (kU/g) Normal control 3.17 ±0.56 1.11 ±0.20 039 ±0.06 Model group 1.20±0.15 d 3.20±0.54 d 0.84±0.79 d Sham operation group 3.16±0.58 1.09 + 0.20 0.41 ±0.04 genetically engineered plasmin
1.71 ±0.24af 3.20 ±0.38 0.75 ±0.151.71 ±0.24 af 3.20 ±0.38 0.75 ±0.15
( 2500U/kg ) 基因工程纤溶酶 (2500U/kg) genetically engineered plasmin
2.20 ± 0.18be 2.80±0.35a 0.74土 0.09a ( 5000U/kg) 2.20 ± 0.18 be 2.80 ± 0.35 a 0.74 soil 0.09 a ( 5000U/kg)
与 支手术组比较， d: p < 0.001; 与模型组比较， a: p <0.05, b: p <0.01; 与模型 +基因工程纤溶酶 5000U/kg 组比较， e: p < 0.05, , f: ρ < 0.01ο 上述实验结果表明基因工程纤溶酶可缩小梗死面积、 减轻脑 水肿和缺血后脑组织总抗氧化能力的降低， 减低 NO含量、 降低 NOS活性， 故对脑缺血起到保护作用。 实施例 12. 基因工程纤溶酶蛋白制剂的应用——溶栓作用 家兔颈外静脉血栓法操作筒便， 不需特殊仪器， 在国内外均 有研究室采用， 是研究药物溶血栓作用的可行方法。 该方法可参 考 [赵炜明,许超千,何树庄，王 玲，基因工程纤溶酶溶栓作用的实 险研究： 哈尔滨医科大学学才艮， 2002 ( 36 ) 3 : 183-185] [徐叔云， 卞如濂，陈修主编.药理实验方法学.北京：人民卫生出版 社, 1982.] [朱燕， 何执中， 孙可迪， 王少飞， 基因工程纤溶酶对实 一除性血栓的影响， 中国药科大学学 4艮， 2000,31(1):50 ~ 52。 ] Compared with the operation group, d: p <0.001; compared with the model group, a: p < 0.05, b: p <0.01; compared with the model + genetically engineered plasmin 5000 U/kg group, e: p < 0.05, f: ρ < 0.01ο The above experimental results show that genetically engineered plasmin can reduce the infarct size, reduce the cerebral edema and reduce the total antioxidant capacity of brain tissue after ischemia, reduce the NO content and reduce the activity of NOS, so it has a protective effect on cerebral ischemia. Example 12. Application of genetically engineered plasmin protein preparation - thrombolytic effect Rabbit external jugular vein thrombosis operation tube, no special equipment, used in research laboratories at home and abroad, is to study drug thrombolytic effect Possible method. This method can be referred to [Zhao Yuming, Xu Chaoqian, He Shuzhuang, Wang Ling, the risk study of genetic engineering fibrinolytic enzyme thrombolysis: Harbin Medical University, Xuecai, 2002 ( 36 ) 3 : 183-185] [Xu Shuyun, Ru Ruyu, Chen Editor-in-Chief. Pharmacological Experimental Methodology. Beijing: People's Medical Publishing House, 1982.] [Zhu Yan, He Zhizhong, Sun Kedi, Wang Shaofei, The effect of genetically engineered plasmin on real destructive thrombosis, 4 years in Chinese Pharmaceutical University, 2000, 31 (1): 50 ~ 52. ]
( 1 ) 制造颈动-静脉旁路实险模型 将实验兔用 20%乌拉坦 5ml/ kg耳缘静脉注射麻醉,背位固定， 剥离气管,插入一塑料套管（气管分泌物多时可通过此套管吸出）, 分离左侧颈外静脉和右侧颈总动脉。 在三段聚乙烯管中段放入一 段长 6cm 的 4 号手术丝线,聚乙烯管内充满肝素生理盐水溶液 ( 5 OU/ml)：用聚乙烯管的一端插入左颈外静脉后，由聚乙烯管准确 注入肝素生理盐水溶液 (50U/ml)抗凝,然后再将聚乙烯管的另一 端插入右颈总动^ ,打开动 夹,血液从右颈总动^流至聚乙烯管 内，返回左颈外静脉,形成颈动-静脉旁路，维持 3 h后，取血,中断血 流,迅速取出丝线称重。 (1) Manufacture of the neck-vein bypass risk model. The rabbits were anesthetized with 20% urethane 5ml/kg ear vein, the back position was fixed, the trachea was peeled off, and a plastic cannula was inserted (the tracheal secretion can be used for a long time) The cannula is aspirated), separating the left external jugular vein and the right common carotid artery. Place a 6cm long surgical wire in the middle of the three-stage polyethylene tube. The polyethylene tube is filled with heparin saline solution (5 OU/ml): one end of the polyethylene tube is inserted into the left external jugular vein and the polyethylene tube is used. Accurately inject heparin physiological saline solution (50U/ml) anticoagulation, then insert the other end of the polyethylene tube into the right neck total motion ^, open the movable clamp, and the blood flows from the right neck to the polyethylene tube. Inside, return to the left external jugular vein to form a carotid-venous bypass. After 3 hours, the blood was taken, the blood flow was interrupted, and the silk wire was quickly taken out for weighing.
( 2 ) 实验分组及给药途径 家兔 36只， 体重 2.5士 0.2kg, 随机分成 6组， 分别为模型 组、 生理盐水对照组、 基因工程纤溶酶小剂量组、 基因工程纤溶 酶中剂量组、 基因工程纤溶酶大剂量组、 尿激酶阳性对照组。 各组具体给药途径如下： 模型组： 所制造的颈动 -静脉旁路模 型； 生理盐水对照组： 耳缘静脉等体积注射生理盐水； 基因工程 歼溶酶小剂量组： 耳缘静脉注射基因工程纤溶酶 1250U/kg; 基因 工程纤溶酶中剂量组： 耳缘静脉注射基因工程纤溶酶 2500U/kg; 基因工程纤溶酶大剂量组： 耳缘静脉注射基因工程纤溶酶 5000U/kg;尿激酶阳性对照组：耳缘静脉注射尿激酶 2 X 104U/kg。 给药方法： 颈动-静脉旁路模型建立后，生理盐水对照组、 基 因工程纤溶酶小剂量组、 中剂量组、 大剂量组及尿激酶阳性对照 组分别于旁路建立后 15min给药， 观察 3h 。 (2) 36 groups of rabbits, weighing 2.5 士 0.2 kg, were randomly divided into 6 groups, which were model group, saline control group, genetically engineered plasmin low-dose group, and genetically engineered plasmin. The dose group, the genetically engineered plasmin high-dose group, and the urokinase-positive control group. The specific administration routes of each group were as follows: 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.
( 3 ) 溶栓作用 采用兔动-静脉旁路血栓形成模型 ,测定 蚓纤溶酶的溶栓作 用 （血栓湿重、 干重）； 心脏取血， 测定丘蚓纤溶酶对血浆纤维 蛋白酶（FIB )、 血浆优球蛋白溶解时间 （ELT )、 纤维蛋白裂解产 物 （FDP ) 等生化指标的影响。 实验结果见表 6和表 7。 本实验中基因工程纤溶酶中剂量组 与大剂量组可使 ELT显著缩短，说明基因工程纤溶酶激活了纤溶 系统。 本实验中基因工程纤溶酶小剂量组、 中剂量组和大剂量组 与模型组相比可明显增加 FDP含量，说明基因工程纤溶酶可使血 液系统的纤溶活性增强。 在本实险中， FIB没有明显的变化,表明 基因工程纤溶酶不易产生出血的副反应。 (3) thrombolysis using a rabbit arteriovenous bypass thrombosis model to determine the thrombolytic effect of sputum fibrinolytic enzyme (thrombosis, dry weight); blood sampling from the heart, determination of quinox fibrinolytic enzyme on plasma fibers Effects of biochemical indicators such as protease (FIB), plasma euglobulin lysis time (ELT), and fibrin cleavage product (FDP). The experimental results are shown in Tables 6 and 7. In this experiment, the genetically engineered plasmin medium-dose group and the high-dose group can significantly shorten the ELT, indicating that the genetically engineered plasmin activates the fibrinolytic system. In this experiment, 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. In this real risk, there was no significant change in FIB, indicating that genetically engineered plasmin was less prone to bleeding side effects.
表 6 基因工程纤溶酶对 ETL FDP数值及 FIB含量的影响 Table 6 Effect of genetically engineered plasmin on ETL FDP and FIB content
ELT ( min ) FDP ( ng · L-1) ELT ( min ) FDP ( ng · L- 1 )
组 另 'J > FIB ( g · L"1 ) Group another 'J > FIB ( g · L" 1 )
< 120 > 130 <5 5-20  < 120 > 130 <5 5-20
20 模型组 0 7.9 8.1 0 0 4.60 ±2.89 生理盐水组 0 7.9 8.1 0 0 5.90 ±2.98 基因工程纤溶  20 Model group 0 7.9 8.1 0 0 4.60 ±2.89 saline group 0 7.9 8.1 0 0 5.90 ±2.98 Genetically engineered fibrinolysis
醉小剂量组 0 7.8 4.9 0 2.5 5.34±2.80ώ Drunk small dose group 0 7.8 4.9 0 2.5 5.34±2.80 ώ
( 1250U/kg ) ( 1250U/kg )
基因工程纤溶 Genetic engineering fibrinolysis
醉中剂量组 4.1 ' 4.1' 1.1 4· 3' 3.75 ± 1.84*Drunk middle dose group 4.1 ' 4.1' 1.1 4· 3' 3.75 ± 1.84*
( 2500U/kg ) ( 2500U/kg )
基因工程纤溶 Genetic engineering fibrinolysis
酶大剂量组 5' 3' 1 1 6 3.00+ 1.93Enzyme high dose group 5' 3' 1 1 6 3.00+ 1.93
( 5000U/kg ) 尿激酶组 5' 3. 0 1 7' 2.60 ± 1.02* ( 5000U/kg ) urokinase group 5' 3. 0 1 7' 2.60 ± 1.02*
ELT, FDP均以例数计， 经秩和检 , .与生理盐水组比较， 均为 p <0.01;FIB含量经 q检验，☆与生理盐水组比较 p <0.05。 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.
表 7 基因工程纤溶酶对家兔体内血栓形成的影响 （ X ±S) 组 另 U 例 剂量 湿重 （ng) 干重 （ng) 抑 制 数 率 (％) 模型组 8 70.25 +2.04 22.01土 1·83 Table 7 Effect of genetic engineering plasmin on thrombosis in rabbits (X ± S) Group U dose Dose (ng) Dry weight (ng) Inhibition rate (%) Model group 8 70.25 +2.04 22.01 Soil 1 ·83
重理盐 8 85.31 ±3.61 28.25 + 1.02 Relieving salt 8 85.31 ±3.61 28.25 + 1.02
水组 基因工 8 1250U/kg 63.58 ±9.60ΔΔ 19.55 ±3.67ΔΔ 24.5 程纤溶 Water group genetic engineering 8 1250U/kg 63.58 ±9.60 ΔΔ 19.55 ±3.67 ΔΔ 24.5 Cheng fibrinolysis
酵小剂 Yeast small
量组 基因工 8 2500U/kg 39.00±5.9··Δ 14.26 ± 1.05 · Δ Δ* 53.6 程纤溶 Volume group genetic engineering 8 2500U/kg 39.00±5.9·· Δ 14.26 ± 1.05 · Δ Δ * 53.6 Cheng fibrinolysis
酶中剂 Enzyme medium
量组 基因工 8 5000U/kg 29.49土 4.58 ' ΔΔ 12.21 ±2.0Γ·Δ Δ 65.3 程纤溶 Volume group genetic engineering 8 5000U/kg 29.49 soil 4.58 ' ΔΔ 12.21 ±2.0Γ· Δ Δ 65.3 Cheng fibrinolysis
酶大剂 Enzyme big agent
量组 Volume group
尿激酶 8 2 x 104U 21.11 ±5·48··ΔΔ 8.91 ±2.07 ΔΔ 75.1 组 /kg Urokinase 8 2 x 10 4 U 21.11 ±5·48·· ΔΔ 8.91 ±2.07 ΔΔ 75.1 group/kg
• ·与模型组比较， ρ < 0.01; Δ△与生理盐水组比较 ρ < ☆大、 中、 小剂量组比较； ρ <0.05。 虽然本文通过优选实施方案详细地演示说明了本发明， 但是 本领域的技术人员知道， 在没有背离本发明的精神和范围的基础 上， 可有各种细节上和形式上的变通。 仅使用常规实脸可得到的 对本发明各种变通， 以及等同效果的各种方案都包括在本申请的 权利要求书的范围内。 • Compared with the model group, ρ <0.01; Δ△ compared with the saline group ρ < ☆ large, medium and small dose groups; ρ <0.05. While the invention has been described in detail herein with reference to the preferred embodiments of the embodiments of the invention Various modifications of the invention, and equivalents thereof, which are available to the present invention, are intended to be included within the scope of the appended claims.

Claims

权利要求书 Claim
1. 一种蚯蚓纤溶酶基因， 其特征在于包含 SEQ ID NO: 1所示的 核苷酸序列。 A limulus fibrinolytic enzyme gene comprising the nucleotide sequence shown in SEQ ID NO: 1.
2. 一种权利要求 1的蚯蚓纤溶酶基因编码的蚯蚓纤溶酶蛋白， 其 特征在于包含 SEQ ID NO: 2所示的氨基酸序列。 A purine plasmin protein encoded by the limulus plasmin gene of claim 1, which comprises the amino acid sequence of SEQ ID NO: 2.
3. 根据权利要求 2所述的蚯蚓纤溶酶蛋白， 其特征在于所述蛋白 盾的分子量为 26.4KD, 等电点为 4.5。 3. The limulus plasmin protein according to claim 2, wherein the protein shield has a molecular weight of 26.4 KD and an isoelectric point of 4.5.
4. 一种制备权利要求 1的蚯蚓纤溶酶基因的方法， 其特征在于包 括下列步骤： A method of producing the limulus plasmin gene of claim 1, which comprises the steps of:
A. 扩增引物合成； A. amplification primer synthesis;
B. 丘蚓总 RNA提取；  B. Total RNA extraction from the hillock;
C. 反转录合成 cDNA的第一条链；  C. reverse transcription synthesis of the first strand of cDNA;
D. 目的基因 PCR扩增；  D. target gene PCR amplification;
E. 目的基因片段的回收；  E. recovery of the target gene fragment;
F. 目的基因片段克隆入载体。 F. The target gene fragment was cloned into a vector.
5. 一种毕赤酵母基因工程菌株， 其特征在于所述菌林能表达蚯蚓 纤溶酵。 A Pichia pastoris genetically engineered strain characterized in that the fungus forest expresses fibrinolytic activity.
6. 根据权利要求 5所述的毕赤酵母基因工程菌株， 其中特征在 于所述菌株毕赤酵母 Pichia pastoris X-33 ( pPIC 6a A-EFE ), CCTCC M204005。 The Pichia pastoris genetically engineered strain according to claim 5, wherein the strain Pichia pastoris X-33 (pPIC 6a A-EFE), CCTCC M204005.
7. 根据权利要求 5所述的毕赤酵母基因工程菌株， 其中特征在 于所述菌株毕赤酵母为毕赤酵母 Pichia pastoris X-33 ( pPIC 6A-EFE ), CCTCC M204004。 The Pichia pastoris genetically engineered strain according to claim 5, wherein the Pichia pastoris is Pichia pastoris X-33 (pPIC 6A-EFE), CCTCC M204004.
8. 一种构建权利要求 5 , 6和 7 中任何一项的毕赤酵母基因工 程菌株的方法， 其特征在于包括： a) 蚓纤溶酶基因的 PCR扩增； b) PCR产物克隆至 T载体； c) 穿梭表达质粒的构建; A method of constructing a Pichia pastoris genetically engineered strain according to any one of claims 5, 6 and 7, which comprises: a) PCR amplification of a sputum plasmin gene; b) cloning of the PCR product to T Vector; c) construction of a shuttle expression plasmid;
d) 表达纤溶酶基因的酵母基因工程菌株的构建。  d) Construction of a yeast genetically engineered strain expressing a plasmin gene.
9. 一种治疗心脑血管疾病的药物， 其特征在于所述药物包括基 因工程蚯蚓纤溶酶蛋白。 A medicament for treating cardiovascular and cerebrovascular diseases, characterized in that the medicament comprises a genetically engineered fibrinolytic protein.
10. 根据权利要求 9的药物， 其特征在于所述药物进一步包括药 学上可接受的载体和 /或其他药物。 10. Medicament according to claim 9, characterized in that the medicament further comprises a pharmaceutically acceptable carrier and/or other medicament.
11. 一种治疗与血液流变学相关的疾病的药物， 其特征在于所述 药物包含基因工程丘蚓纤溶酶蛋白。 A medicament for treating a disease associated with hemorheology, characterized in that the medicament comprises genetically engineered 蚓 蚓 plasmin protein.
12. 根据权利要求 11 的药物， 其特征在于所述药物进一步包括 药学上可接受的载体， 和 /或其他药物。  12. Medicament according to claim 11, characterized in that the medicament further comprises a pharmaceutically acceptable carrier, and / or other medicament.
13. 一种治疗栓塞性心脑血管疾病的药剂， 其特征在于所述药剂 包含基因工程丘蚓纤溶酶蛋白。  13. An agent for treating embolic cardiovascular and cerebrovascular diseases, characterized in that the agent comprises genetically engineered mound plasmin protein.
14. 才艮据权利要求 13 的药剂， 其特征在于所述药剂进一步包括 药学上可接受的载体， 和 /或其他药物。  14. The agent according to claim 13, characterized in that the agent further comprises a pharmaceutically acceptable carrier, and/or other drug.
15. 根据权利要求 13和 14中任何一项的药剂， 其中所述药剂为 口服药剂。  The agent according to any one of claims 13 and 14, wherein the agent is an oral agent.
16. 基因工程蚯蚓纤溶酶蛋白在制备治疗心脑血管疾病的药物 中的应用。  16. Genetic engineering 蚯蚓 plasmin protein for the preparation of drugs for the treatment of cardiovascular and cerebrovascular diseases.
17. 基因工程蚯蚓纤溶酶在制备预防血栓疾病、 有益心血管健康 的保健品中的应用。  17. Genetic engineering 蚯蚓 plasmin in the preparation of health products for the prevention of thrombotic diseases and cardiovascular health.
PCT/CN2005/000501 2004-04-14 2005-04-14 Earthworm fribrinolytic enzyme gene, genetic engineering strains, and the construction and usages thereof WO2005100570A1 (en)

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CN 200410034474 CN1259411C (en) 2004-04-14 2004-04-14 Strain of Pichia yeast genetic engineering, preparation method and application
CN 200410034475 CN1563387A (en) 2004-04-14 2004-04-14 Gene of earthworm plasmin and genetic engineerng strain, construction and application
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