WO1995027050A1 - Procede de preparation de streptokinase recombinee - Google Patents

Procede de preparation de streptokinase recombinee Download PDF

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Publication number
WO1995027050A1
WO1995027050A1 PCT/CN1995/000024 CN9500024W WO9527050A1 WO 1995027050 A1 WO1995027050 A1 WO 1995027050A1 CN 9500024 W CN9500024 W CN 9500024W WO 9527050 A1 WO9527050 A1 WO 9527050A1
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WO
WIPO (PCT)
Prior art keywords
streptokinase
expression
gene
bacteria
plasmid
Prior art date
Application number
PCT/CN1995/000024
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English (en)
Chinese (zh)
Inventor
Houyan Song
Original Assignee
Shanghai Medical University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Medical University filed Critical Shanghai Medical University
Priority to CH03481/95A priority Critical patent/CH688653A5/de
Priority to RU96121788/13A priority patent/RU2127758C1/ru
Publication of WO1995027050A1 publication Critical patent/WO1995027050A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3153—Streptokinase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention belongs to the field of biochemistry. More specifically, the present invention relates to a method for preparing a recombinant streptokinase. Background technique
  • thrombolytic drugs include tissue plasminogen activator (rt-PA), streptokinase (SK), and benzoyl plasminogen streptokinase complex (APSAC).
  • rt-PA tissue plasminogen activator
  • SK streptokinase
  • APSAC benzoyl plasminogen streptokinase complex
  • the purpose of the present invention is to provide a method for preparing recombinant streptokinase by using biological high technology.
  • the preparation method of the method is safe, the product yield and purity are high, and the cost is low.
  • a method for preparing recombinant streptokinase is characterized in that:
  • primer used in the present invention refers to a single-stranded DNA oligonucleotide sequence, which can be used as a starting point for the synthesis of an extension, and the primer is partially or completely complementary to the nucleic acid strand to be amplified.
  • the length and order of the primers must be such that they can initiate the synthesis of extension products.
  • the primers are about 5-50 nucleotides. The specific length and sequence depend on the complexity of the desired target DNA or RNA, and the conditions under which the primers are used, such as temperature and ionic strength.
  • vector may include a plasmid, a cosmid end plasmid, a phage, or a virus.
  • the vector is a prokaryotic expression plasmid. The PCR amplified product was cloned into the appropriate site of the expression vector to form a highly efficient expression vector.
  • the expression vector can be introduced into an appropriate host cell by any of a series of suitable methods, such as by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, and the like.
  • the host cells used in the present invention may be prokaryotic.
  • Ideal prokaryotic hosts include bacteria of the genus Escherichia coli, B. subtilis, and the like. The most ideal prokaryotic host is E. coli.
  • the host cells are grown on a selection medium, and cells containing a highly efficient expression vector are selected.
  • the host cell containing the high-efficiency expression vector After screening the host cell containing the high-efficiency expression vector, it can be cultured under conditions suitable for host cell growth and the appropriate conditions can be used to induce the expression of the streptokinase gene.
  • the expression of a streptokinase gene is induced by heat.
  • the gene expression product of streptokinase can be isolated and purified by isolation and purification methods well-known to those skilled in the art to obtain a purified expression product.
  • the table Tat products exist in the form of inclusion bodies in the host cell, which can simplify the purification process and improve product purity. Detailed description of the invention
  • the present invention uses recombinant DNA technology to efficiently express r- in non-pathogenic E. coli SK, the method is safe and does not cause any harm to the environment and operators. Due to its simple expression and purification methods, the purity and yield of the product are very high, thus greatly reducing the production cost and price of r-SK.
  • Figure 1 shows the construction of plY-4 plasmid
  • Figure 2 is a construction diagram of a streptokinase gene expression plasmid.
  • the sequence of the PCR nucleotide primers was designed according to the sequence of 5 amino acids each at the C-terminus of the N-terminus of streptokinase.
  • the PCR products obtained with the following primers were specific bands.
  • the specific primer sequence is as follows:
  • the two primers were synthesized by a DNA synthesizer using the solid-phase phosphate triester method and purified for PCR amplification of the streptokinase gene.
  • the 5 'ends of the two PCR primers each have a restriction enzyme recognition sequence.
  • Macromolecular DNA of No. 8 Streptococcus was prepared according to the method of Frederick M. Ausubel et al, and A 26 was used . / A 28 . The ratio is> 2. 0.
  • the PCR reaction system and conditions are as follows:
  • Tag DNA polymerase lul (2. 5U) Denaturation temperature 90 ° C for 60 seconds, extension temperature 72 ° C for 120 seconds, annealing 52'C for 60 seconds, add 2.5 UTag enzyme after 15 cycles, and continue for 15 cycles . Take 2ul of the reaction and identify it by agarose gel electrophoresis. A single band of 1.3Kb is observed, which is consistent with the length of the complete streptokinase gene. The reaction was hydrolyzed by endonucleases and identified by agarose gel electrophoresis, showing characteristic endonuclease sites in the streptokinase gene.
  • the above PCR reaction product and pUC19 plasmid were each completely hydrolyzed by the same two endonucleases, and then added to the T4 DNA ligase reaction system, left at room temperature for 1 hour, transformed into E. coli JM83, and screened in a 37'C incubator with ampicillin-LB plate to cultivate.
  • plasmid A single colony was taken to prepare a plasmid, and the plasmid was hydrolyzed with several endonucleases, respectively, and identified by agarose electrophoresis, and the plasmid showing the characteristic band of SK gene was named (pST-1).
  • pST-1 plasmid nucleotide sequence analysis Because the SK gene contains Hind I cleavage points, two subclones of 770bp and 570bp fragments were first constructed, and then a nucleotide sequence analysis was performed to confirm the nucleotide sequence of the streptokinase gene and Reading frame. The measured results are shown in Table 1.
  • the pST-1 plasmid was hydrolyzed with restriction enzymes, separated by agarose gel electrophoresis, and the 1.3 kb SK gene was recovered, and then recombined with the prokaryotic expression plasmid vector.
  • the ligation reaction was transformed into E. coli K802 by the calcium phosphate method, and was screened and cultured by ampicillin. Pick a single colony, prepare a plasmid, and identify it with the restriction enzyme fragment length. Those that contain a characteristic fragment of the SK gene are called pSTE-1 expression plasmids, and bacteria containing the pSTE-1 expression plasmid are called engineering bacteria PSTE-1.
  • Sample solution Contains 0.025M Tris-HCl (pH 6.7), 2% SDS, 10% glycerol, 5% mercaptoethanol, and 0.001% bromophenol blue.
  • Sample processing and spotting After 1ml of induction culture, the bacterial solution was centrifuged and the precipitate was suspended in 100ul sample dissolution solution, and placed in a boiling water bath for 5 minutes to dissolve the precipitate. Samples of chromatographic peaks were mixed with an equal volume of 2 times the concentration of the sample dissolving solution and then heat-treated, and the spotting amounts were 20ug each. Samples of control bacteria or bacteria before induction were cultured in the same way, and samples were treated under the same conditions.
  • Expression level Stained bands of proteins in the gel were scanned with Shimadzu CS-910 dual wavelength The instrument performs blackness scanning and computer processing to print the percentage of r -SK peak.
  • the state of the expression product in the bacterial cell-the bacterial cell was broken by ultrasound and a high-pressure homogenizing pump. After centrifugation, the supernatant and the pellet containing the same amount of protein were spotted separately and subjected to SDS-PAGE. The results showed that in the row of the precipitated lysate, the staining band was deep at the molecular weight of 43,000, and the spotting was the supernatant. In the same row of the liquid, almost no staining was observed, indicating that the r-SK expression product formed inclusion bodies.
  • NBS BIO Fill Tank Fermentation-A single colony of PSTE-1 was cultured in 100ml LB-Amp medium at 30'C overnight, and A 6Q was measured. As a seed liquid for fermentation.
  • the bacterial solution is stored after centrifugation or used for secondary fermentation.
  • LB broth Same as above except that Agar and Amp are not added.
  • the H 2 0 in the tank is released, and the culture medium is replaced, and various parameters are set.
  • take out the secondary seed bacteria inoculate the fermenter at 1:10, ventilate it for fermentation.
  • the following conditions are maintained: temperature: 30'C, pH: 6-8, DO: 50-80%, stirring speed: DO-based automatic control, 5ml of bacterial solution is taken out every hour to check the bacterial density
  • Bacteria Wet bacteria are suspended in a buffer solution, added to a high-pressure homogenizer pump, and the bacteria are burst with an appropriate pressure.
  • Quality control index Use a microscope to check the degree of bacterial fragmentation. Inspection method: The bacterial suspension before homogenization is smeared after appropriate dilution, Gram staining, microscopic examination of the number of bacteria in 4 fields (A), homogenate After the suspension was diluted similarly, smear was performed uniformly, and the number of unbroken bacteria in 4 visual fields was observed (B).
  • Bacteria breaking rate% (A— B) / AX 100%
  • the precipitate was collected by centrifugation, that is, the crude inclusion body, in which the r-SK accounted for more than 75-85% of the total bacterial protein.
  • the purified inclusion bodies were dissolved with 6M guanidine hydrochloride, centrifuged at 1000 rmp for 30 minutes, and the supernatant was removed.
  • the PB was dialyzed several times, centrifuged at 20000 rmp for 30 minutes, and the supernatant was left. At this time, r-SK accounted for about 95% of the bacterial protein.
  • the activity is above 65 thousand IU / mg, and the r-SK yield is about 65%.
  • Ion exchange chromatography Waters650 or Econo chromatography system.
  • the anion exchange column was equilibrated with PB, and the refolded r-SK solution was passed through the column at a flow rate of 1.0 ml / min , and then the column was washed with PB, and the linear pH gradient of PB was used to elute r-SK, the flow rate: 4. Oml / min, collected The first elution peak (r-SK).
  • Sterile filtration Under the condition that the cleanliness reaches level 100, it is filtered with a 0.22 ⁇ sterile filter.
  • the packed samples are placed in a lyophilizer and lyophilized, and the stopper is automatically added in the lyophilizer, and then the aluminum cap is labeled, labeled, and boxed.
  • Angiography proved that r-SK has a significant effect on dog femoral artery thrombosis and dog coronary artery thrombosis.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne la construction du plasmide d'expression pSTE-SK-1 à l'aide de vecteurs de streptokinase, par exemple le PLY-4, et la transformation de E. coli à l'aide dudit plasmide, l'expression du gène r-SK étant induite par chauffage. Les bactéries manipulées sont amplifiées par fermentation, détruites, le corps d'inclusion est récupéré par centrifugation, et le r-SK est purifié selon un procédé à deux étapes après renaturation. Le rendement du procédé et la pureté obtenue par ce procédé sont élevés et le coût est faible.
PCT/CN1995/000024 1994-04-04 1995-04-03 Procede de preparation de streptokinase recombinee WO1995027050A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CH03481/95A CH688653A5 (de) 1994-04-04 1995-04-03 Verfahren zur herstellung rekombinanter streptokinase
RU96121788/13A RU2127758C1 (ru) 1994-04-04 1995-04-03 Способ получения рекомбинантной стрептокиназы

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN94112106.2 1994-04-04
CN 94112106 CN1035193C (zh) 1994-04-04 1994-04-04 一种制备重组链激酶的方法

Publications (1)

Publication Number Publication Date
WO1995027050A1 true WO1995027050A1 (fr) 1995-10-12

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CN (1) CN1035193C (fr)
CH (1) CH688653A5 (fr)
RU (1) RU2127758C1 (fr)
WO (1) WO1995027050A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1064406C (zh) * 1998-05-11 2001-04-11 中国人民解放军军事医学科学院微生物流行病研究所 利用基因工程技术生产葡激酶的方法
AR071169A1 (es) * 2008-03-31 2010-06-02 Council Scient Ind Res Variantes de la estreptoquinasa que contienen cisteina y sus formas covalentemente modificadas
CN102936603A (zh) * 2012-10-31 2013-02-20 上海昊海生物科技股份有限公司 尿苷二磷酸-葡萄糖脱氢酶的表达及酶活测定

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0151337A2 (fr) * 1983-10-10 1985-08-14 The Board of Regents for the University of Oklahoma Vecteurs recombinants codant pour la streptokinase
EP0407942A2 (fr) * 1989-07-11 1991-01-16 Otsuka Pharmaceutical Factory, Inc. Protéines de streptokinase, les gènes correspondants, les plasmides correspondants et procédé pour leur préparation
US5066589A (en) * 1984-03-02 1991-11-19 Board Of Regents Of The University Of Okla. Streptokinase-coding recombinant vectors
EP0489201A1 (fr) * 1990-05-23 1992-06-10 Centro De Ingenieria Genetica Y Biotecnologia Procédé d'isolement d'expression d'un gène codant pour la steptokinase, séquence nucléotidique obtenue, ADN recombinant et microorganisme transformé

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0151337A2 (fr) * 1983-10-10 1985-08-14 The Board of Regents for the University of Oklahoma Vecteurs recombinants codant pour la streptokinase
US5066589A (en) * 1984-03-02 1991-11-19 Board Of Regents Of The University Of Okla. Streptokinase-coding recombinant vectors
EP0407942A2 (fr) * 1989-07-11 1991-01-16 Otsuka Pharmaceutical Factory, Inc. Protéines de streptokinase, les gènes correspondants, les plasmides correspondants et procédé pour leur préparation
EP0489201A1 (fr) * 1990-05-23 1992-06-10 Centro De Ingenieria Genetica Y Biotecnologia Procédé d'isolement d'expression d'un gène codant pour la steptokinase, séquence nucléotidique obtenue, ADN recombinant et microorganisme transformé
US5296366A (en) * 1990-05-23 1994-03-22 Centro De Ingenieria Genetica Y Biotecnologia Method for the isolation and expression of a gene which codes for streptokinase, nucleotide sequence obtained, recombinant DNA and transformed microorgnaisms

Also Published As

Publication number Publication date
CN1035193C (zh) 1997-06-18
CN1096326A (zh) 1994-12-14
RU2127758C1 (ru) 1999-03-20
CH688653A5 (de) 1997-12-31

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