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WO1995027048A1 - Procede de preparation de staphylokinase recombinee - Google Patents

Procede de preparation de staphylokinase recombinee Download PDF

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Publication number
WO1995027048A1
WO1995027048A1 PCT/CN1995/000025 CN9500025W WO9527048A1 WO 1995027048 A1 WO1995027048 A1 WO 1995027048A1 CN 9500025 W CN9500025 W CN 9500025W WO 9527048 A1 WO9527048 A1 WO 9527048A1
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WO
WIPO (PCT)
Prior art keywords
sak
gene
expression vector
expression
staphylokinase
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/CN1995/000025
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English (en)
Chinese (zh)
Inventor
Houyan Song
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Medical University
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
Publication of WO1995027048A1 publication Critical patent/WO1995027048A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)

Definitions

  • the present invention relates to the field of biotechnology, and more particularly, to a method for preparing recombinant staphylokinase by using biotechnology. Background technique
  • Staphylokinase is a proteolytic enzyme synthesized by lysogenic phage of Staphylococcus aureus. Staphylokinase has a molecular weight of about 15.5KD.
  • the process of activating plasminogen (pig) is similar to streptokinase. First SAK and pig combine to form a 1: 1 complex, and then the complex activates another molecular pig to form Plasmin, which catalyzes the hydrolysis of fibrin, dissolves blood clots and restores blood circulation.
  • the object of the present invention is to provide a new method for preparing recombinant staphylokinase, which has a simple purification method, high product purity, high yield, and low cost.
  • the invention provides a method for preparing recombinant staphylokinase (r-S A K):
  • primer refers to a single-stranded DNA oligonucleotide sequence that can serve as a starting point for the synthesis of an extension that 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 depends on the desired target DNA or
  • RNA The complexity of the 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. PCR amplification product cloned into the appropriate site of the expression vector 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 cell used in the present invention may be a cell of a prokaryotic or eukaryotic organism.
  • Ideal prokaryotic hosts include bacteria of the genus Escherichia coli, B. subtilis, and the like. The most ideal prokaryotic host is E. coli. The ideal eukaryotic host is yeast.
  • the host cells are grown on a selection medium, and cells containing the expression vector are selected.
  • the host cell containing the expression vector After the host cell containing the expression vector is selected, it can be cultured under conditions suitable for the growth of the host cell, and the expression of the staphylokinase gene can be induced under appropriate conditions. Preferably, the expression of staphylokinase is induced by heat.
  • the staphylokinase gene expression product 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 r- SAK gene is recombined with a prokaryotic expression vector (such as a vector containing PL, PR promoter, CIt857 gene, 5sRNA termination signal and other regulatory elements) to form an expression vector, transform E. coli, and select transformed E. coli (using As engineering bacteria), the expression of r-SAK gene is induced by temperature; the expression product r-SAK exists in the cytoplasm in a soluble and active state, accounting for more than 40% of the bacterial protein.
  • a prokaryotic expression vector such as a vector containing PL, PR promoter, CIt857 gene, 5sRNA termination signal and other regulatory elements
  • Fermented cultured transformed E. coli in which transformed E. coli is cultured with a low nutrient solution, such as pSTE-SAK-1, and fed with LB, glucose, and rare element solution before and after temperature induction.
  • a low nutrient solution such as pSTE-SAK-1
  • LB low nutrient solution
  • glucose and rare element solution before and after temperature induction.
  • the microporous membrane was filtered to sterilize and freeze-dried into a white powdery finished product.
  • Figure 1 shows the construction of the expression vector PLY-4.
  • Figure 2 shows the construction of the expression plasmid pSTE-SAK.
  • the results of N- and C-terminal amino acid sequence determination were used to design PCR primers.
  • the ester method was synthesized by a DNA synthesizer, and was purified to amplify the staphylokinase gene.
  • the 5 'end of each PCR primer pair contains a restriction enzyme recognition site.
  • Primer I 3 '- ⁇ ' reverse primer
  • a very bright specific band appears between 400 and 500 bp, and the length of the nucleic acid fragment contained in it matches the length of the complete staphylokinase gene.
  • the gene amplified by PCR was recombined with the vector PUC-18 or 19 to form a recombinant plasmid.
  • the nucleotide sequence of the staphylokinase gene was determined by DNA nucleotide analysis, and the amino acid sequence was derived from it.
  • the PUC19 plasmid vector and T 4 DNA ligase reaction system which have been completely hydrolyzed by the corresponding enzymes are added. After standing at room temperature for 1 hour, E. coli JM83 is transformed, and ampicillin-LB plates are used. Incubate in a 37 ° C incubator.
  • Sex strippers are named pST- SAK-1,
  • the nucleotide sequence and reading frame of the SAK gene in the plasmid pST-SAK-1 were confirmed by nucleotide sequence analysis.
  • Sample dissolving solution 0.00625M Tris-HCl (pH 6.7), 2% SDS, 10% glycerol, 5% mercaptoethanol, and 0.001% bromophenol blue.
  • Sample processing and spotting 1 ml of induction culture bacterial solution, after centrifugation, the pellet was suspended in 100 ⁇ l sample solution, and placed in a boiling water bath for 5 minutes to dissolve the pellet. Control bacteria or pre-induction bacteria were cultured in the same way and samples were treated under the same conditions. The spot size was 10 ⁇ l, and the amount of protein contained was almost equal.
  • Detection of expression level The Shimadzu CS-910 densitometer was used to scan the gel of SDS-PAGE Coomassie brilliant blue staining, and the results showed that r-SAK accounted for more than 40% of the total protein in the bacteria.
  • the Lowry method was used to determine the protein content.
  • Human thrombin 10 10 10 10 10 10 human fibrinogen
  • Example (2) Large-scale preparation of genetically engineered staphylokinase (r_SAK):
  • the bacteria collection method is the same as that of shake flask culture: the bacteria can be stored at a 20'C for later use.
  • the wet bacteria were washed three times with 1000 ml of PBS (pH 7.4), suspended in PBS (10 gm / 100 ml), squeezed with a high-pressure homogenizer pump, and centrifuged at 15000 r. Pm for 10 minutes to collect the supernatant.
  • the S-Sepharose column was equilibrated with a buffer solution, the above sample was added, and the hybrid protein (3 times the bed volume) was eluted with the buffer solution.
  • the pH gradient and the salt gradient buffer were used to elute r-SAK. Filter and concentrate.
  • the gel column was equilibrated with PBS pH 7.4, and the ultrafiltration concentrated solution was eluted with PBS pH 7.4 to collect and combine each tube solution containing SAK activity. After filtering through 0.22 ⁇ microporous filter membrane, it was aseptically packed and lyophilized into a semi-finished product. After filtering through a microporous filter with a thickness of 0.22 ⁇ m , and adding stabilizers such as human serum albumin, it was aseptically packaged and lyophilized into a finished product.
  • the total protein measured by the Lowry method was 8.21g
  • the total activity measured by the fibrin clot dissolution method was 2.% X 10 8 HU
  • the specific activity was 0.36 X 10 5 HU / mg.
  • the total protein of the supernatant of the press was 7.16g
  • the total activity was 2.93 X 10 8 HU
  • the specific activity was
  • the SAK gene and its regulatory sequence are reported from the phage genome as reported in the off-picture documents or patent documents.
  • the present invention directly uses S. aureus macromolecular DNA as a template to amplify and prepare the SAK gene by PCR.
  • the SAK gene constructed by the present invention uses a large intestine rod
  • the bacteria efficiently express r-SAK, and r-SAK is soluble and active in the bacteria.
  • the purification method is simple and the yield of the product is high. 400-500mg of pure product can be obtained per liter of fermentation broth, and the purity is 97-99%. Compared with the yield of 6 mg per liter of fermentation solution by D. Collen et al., The effect of the present invention is significantly improved, and its cost is much lower.
  • Table 1 lists the protein content, activity, and specific activity during each purification step.
  • the r-SAK prepared by applying the invention has been proved by animal experiments to have good thrombolytic function, such as:
  • r-SAK was used to treat experimental femoral arterial thrombosis in dogs, and angiography showed that the femoral artery was not developed below the treatment before the treatment.
  • R-SAK 0.1 mg / kg was injected intravenously, and the femoral artery was fully filled after 60 minutes. Circulation was restored, and no femoral artery filling was seen in control animals. A total of 8 dogs were made (5 in the treatment group and 3 in the control group).
  • the finished recombinant staphylokinase prepared by the present invention is dissolved in water for injection and used for injection to treat myocardial infarction, pulmonary, brain, peripheral vascular thromboembolism, hemodialysis pathway and blood clotting in blood GTT AT GAAA AAG AAAAA T
  • Trss G A Ls Asn pro lie Tr Glu Lsss Glu Glu Tr Ls Ser Pheh L L Lh

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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)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne la construction du vecteur d'expression pSTE-SAK-1 à l'aide de la staphylokinase amplifiée par PCR et d'un vecteur d'expression procaryote, par exemple le PLY-4, ainsi que la transformation de E. Coli à l'aide de pSTE-SAK-1, l'expression du gène étant induite par chauffage. Les bactéries manipulées sont amplifiées par fermentation, et détruites, puis le r-SAK pur est séparé du surnageant selon un procédé à deux étapes comprenant un échange d'ions et une filtration sur gel. Le rendement du procédé et la pureté de la staphylokinase obtenue par ce procédé sont élevés et le coût est faible.
PCT/CN1995/000025 1994-04-04 1995-04-03 Procede de preparation de staphylokinase recombinee Ceased WO1995027048A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN94112105A CN1035192C (zh) 1994-04-04 1994-04-04 一种重组葡激酶的制备方法
CN94112105.4 1994-04-04

Publications (1)

Publication Number Publication Date
WO1995027048A1 true WO1995027048A1 (fr) 1995-10-12

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CN (1) CN1035192C (fr)
WO (1) WO1995027048A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1059926C (zh) * 1997-07-19 2000-12-27 张其玖 葡激酶基因及其高表达工程菌株
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
CN100342007C (zh) * 2000-01-28 2007-10-10 复旦大学 一种新型重组葡激酶及其制备方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0077664A2 (fr) * 1981-10-16 1983-04-27 Kabushiki Kaisha Yakult Honsha ADN recombinant et bacille coliforme contenant le matériel génétique pour la production de staphylokinase, et la production de staphylokinase
WO1993013209A1 (fr) * 1991-12-30 1993-07-08 Medac Gesellschaft für klinische Spezialpräparate mbH Staphilokinases exemptes de peptides-signal d'expression

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0077664A2 (fr) * 1981-10-16 1983-04-27 Kabushiki Kaisha Yakult Honsha ADN recombinant et bacille coliforme contenant le matériel génétique pour la production de staphylokinase, et la production de staphylokinase
WO1993013209A1 (fr) * 1991-12-30 1993-07-08 Medac Gesellschaft für klinische Spezialpräparate mbH Staphilokinases exemptes de peptides-signal d'expression

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CN1096325A (zh) 1994-12-14
CN1035192C (zh) 1997-06-18

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