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Cell strain and method of expressing reteplase (rPA) by human source cells

A technology of reteplase and cell line, applied in the field of biopharmaceuticals, can solve the problems of low yield, high cost and long cycle

Active Publication Date: 2020-02-21
谢伟全
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, yeast and mammalian cells are used to produce reteplase, which has high cost, long cycle and low yield

Method used

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  • Cell strain and method of expressing reteplase (rPA) by human source cells
  • Cell strain and method of expressing reteplase (rPA) by human source cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Construction of rPA lentiviral expression plasmid

[0061] 1. The complete DNA sequence of rPA was obtained through literature search and analysis, and the DNA sequence (SEQ ID No.2) encoding rPA was obtained by in vitro total gene synthesis by chemical synthesis, and EcoRI (CAS : 1040S, Treasure Bioengineering Co., Ltd.) and NotI enzyme (CAS: 1166S, Treasure Bioengineering Co., Ltd.) sites, and then rPA was recombined into the lentiviral expression vector plenti-puro (CAS: 39481, Addgene ), the correct rPA expression plasmid plenti-rPA-puro was obtained by sequencing.

Embodiment 2

[0063] Construction of rPA expressing cell line

[0064] 1. Preparation of lentiviral particles expressing rPA.

[0065] (1) Pack lentiviral particles according to the 3-plasmid system (pMD2.G, psPAX2, plenti-rPA-puro) (Addgene), and use the 3 plasmids according to a specific ratio (pMD2.G:psPAX2:plenti-rPA-puro=5: 10:15 μg), introduced into 10 by lipofection method (CAS: 40802ES02, Shanghai Yisheng Biotechnology Co., Ltd.) 6 human-derived 293T cells (ATCC), and then put the 293T cells in CO 2 Cultivated in an incubator;

[0066] (2) After culturing for 48 hours, the culture supernatant was collected by centrifugation, 1200 rpm, 5 minutes, an equal amount of culture medium was added to the cells, and the culture was continued;

[0067] (3) After continuing to cultivate for 24 hours, collect the culture supernatant by centrifugation, 1200rpm, 5min;

[0068] (4) The cell culture supernatant collected twice was combined, and 10% volume of PEG8000 / NaCL solution (CAS: A100159-0...

Embodiment 3

[0095] rPA thrombolytic activity assay

[0096] 1. Preparation of fibrin plate: Weigh 0.02g of fibrin (CAS: 9001-32-5, Sigma-Aldrich), add 5ml of PBS, mix and place in a 37°C water bath to preheat until fully dissolved, and take another appropriate amount of coagulation Enzyme (CAS: 9002-04-4, Sigma-Aldrich) was added to 1ml of PBS solution, mixed gently, and placed in a 37°C water bath to preheat; 0.07g of agarose (CAS: 9012-36-6, Sigma-Aldrich) was weighed -Aldrich), add 7ml of PBS solution, heat to dissolve and then cool to about 50°C to obtain an agarose solution; mix the prepared thrombin solution and fibrin solution and immediately add it to the agarose solution, and pour it into the 10cm diameter plates, cooled at room temperature to obtain fibrin plates.

[0097] 2. Use a pipette tip to punch holes in the fibrin plate prepared in Step 1, add 5 μl of the rPA sample prepared in Example 2, and then place it in a 37° C. incubator for 12 hours and observe the size of the t...

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Abstract

The invention belongs to the field of biopharmacy and particularly relates to a method and a cell strain of expressing reteplase by human source cells. The method specifically comprises the followingsteps of genetically synthesizing a cDNA sequence which contains a secretory signal and encodes the reteplase, inserting the cDNA sequence into a lentiviral expression vector, constructing a lentiviral vector capable of expressing the reteplase with secretion, packaging lentiviral particles, infecting the human source cells 293F with the lentiviral particles, performing resistance screeing on a high-copy monoclonal cell strain capable of stably expressing the reteplase with puromycin carried in the lentiviral vector, testing the expression content and the enzymatic activity of the reteplase, and verifying the established expression method. The method and the cell strain have the advantages that the reteplase expression cell strain established by the method is human source 293F cells, doesnot contain pyrogen endotoxin, is suspended, stably expresses the reteplase, is easy in large-scale amplification as well as is less in impurity and easy to purify as expressing the reteplase with secretion.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a new method for secreting and expressing reteplase rPA by human cells. Background technique [0002] At present, thromboembolism is a common cardiovascular disease that seriously endangers human health and brings a heavy burden to patients, families and society. Thrombolytic therapy is currently the most effective means of treating thromboembolic diseases. Reteplase rPA is a better third-generation thrombolytic drug for clinical application. rPA is a deletion mutant of human tissue plasminogen activator tPA. It is a single-chain non-glycosylated protein with a molecular weight of 39 kDa and contains 9 pairs of disulfide bonds. Clinically, rPA is more likely to act on the inside of the thrombus, and has a strong fibrinolytic effect and rapid onset. [0003] In view of the yearly increase of embolism patients, the clinical demand for reteplase is very high. However, the curre...

Claims

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Application Information

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IPC IPC(8): C12N9/72C12N15/867C12N5/10C12R1/91
CPCC12N9/6459C12N15/86C12N2740/15043C12Y304/21068
Inventor 谢伟全
Owner 谢伟全
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