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Preparation method of urokinase

A urokinase and cell technology, applied in the field of preparation of urokinase, can solve the problems of aglycosylation of urokinase, low efficiency of urokinase, difficult purification and the like, and achieve the effects of easy purification, high output and good thrombolytic effect

Inactive Publication Date: 2017-05-17
SHENZHEN BLOT BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In view of this, the present invention provides a method for preparing urokinase, which is used to solve the problems of low efficiency and low purity in extracting urokinase from urine, urokinase extracted from Escherichia coli without glycosylation, and difficult purification

Method used

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  • Preparation method of urokinase
  • Preparation method of urokinase
  • Preparation method of urokinase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The human urokinase gene cDNA was cloned into the vector pMT / Bip / V5-A that can be expressed in Drosophila S2 cells with NcoI and XhoI sites, and 6 histidines were added at the end of the urokinase cDNA cDNA (CATCAT CAC CAT CAC CAT) for easy purification. The N-terminus of the plasmid contains Bip-signal sequence, so that the expressed urokinase can be released from cells for easy purification; it contains ampicillin and puromycin resistance genes, which is convenient for screening. Plasmid information such as figure 1 , figure 2 shown.

Embodiment 2

[0035] Transfect the plasmid obtained in Example 1 into Drosophila S2 cells, the specific operation is: place Drosophila S2 cells in 70-90% transfection mixture, dilute Reagent's transfection reagent culture medium (1:1 ratio dilution), dilute the plasmid in Medium. Add the diluted plasmid to the diluted 2000 reagent, and then incubated for 45min, the mixture was added to the cells.

[0036] 48 hours after transfection, selection was performed with Snyder's medium containing 2 μg / ml puromycin and 50 μg / ml ampicillin in 10% fetal bovine serum. Cells that were not successfully transfected were apoptotically lysed within 1-2 days in the culture medium, and cells that were successfully transfected could survive and be passaged in the culture medium. The medium was changed every four days, and after 3 weeks of selection, Drosophila S2 cells expressing urokinase were obtained.

[0037] The Drosophila S2 cells that can express urokinase were subcultured and expanded. After o...

Embodiment 3

[0039] The interaction between uPA obtained in Example 2 and its receptor uPAR was analyzed by surface plasmon resonance (SPR) in BIAcore 2000 (BIAcore AB, Uppsala, Sweden). uPAR was immobilized on the carboxymethyldextran surface of a CM5 chip (BIAcore AB) using amine coupling according to the manufacturer's instructions. For the fixation procedure, uPAR was diluted to a final concentration of 2.5 μg / ml using sodium acetate buffer, pH 5.5. uPAR was immobilized on the surface for 7 min in separate flow cells at a flow rate of 10 μl / min, and a reference surface was prepared by amine coupling activation followed by immediate deactivation in the remaining two flow cells. For kinetic measurements, six different concentrations of uPA were injected for 3 minutes, followed by a 10-minute dissociation phase. At the end of each cycle, the surface was regenerated by a 1 min injection of 1 M NaCl followed by a 1 min injection of 0.02 M 6-aminocaproic acid, and a brief wash step with pho...

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Abstract

The application belongs to the technical field of biology, and particularly relates to a preparation method of urokinase. The urokinase expressed by the method is high in output and easy to purify; the expressed urokinase proteins glycosylation is similar with the urokinase expressed by human body, and functions are the same. Moreover, the method is free from the limit of natural resources, and there is no limit to produce the urokinase with the same structure as the human body according to market demand; the method is less in investment, high in output, good in thrombolysis effect, and free from toxic side effects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing urokinase. Background technique [0002] Venous thrombosis is one of the main diseases that endanger human health. In my country, there are more than ten million cardiovascular and cerebrovascular cases every year, and the treatment cost is tens of billions. More than one million patients die from thrombotic diseases every year. At present, thrombolytic drugs are mostly used to treat venous thrombotic diseases clinically, and the sales volume of the thrombolytic drug market has reached more than 100 billion US dollars. [0003] Urokinase has been used for decades as a thrombolytic agent in the treatment of venous thrombosis. The urokinase currently used in clinical treatment is directly extracted from urine, and the second is to express recombinant urokinase in Escherichia coli. Urokinase, which is widely used in China, is extracted from fresh hum...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/72C12N15/58C12N15/85
CPCC12N9/6462C12N15/85C12N2800/105C12Y304/21031
Inventor 王海瑶靳海宁熊祖应
Owner SHENZHEN BLOT BIOTECH
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