Cell strain transfacted by delection/Point mutont gene using histo type cellulolytic proenzyme as activator

A technology of plasminogen and gene transfection, which is applied in the field of cell biology and molecular biology, and can solve problems such as difficulty in wide application and increased production costs

Inactive Publication Date: 2005-01-05
NINGXIA UNIVERSITY
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Problems solved by technology

But for rPA, not only the site of action of t-PA rapid inhibitor PAI-1 (plasminogen activator inhibitor -1) still exists on its gene, but also because it is an expression product of Escherichia coli, a large amount of purification is required and renaturation work, thus increasing the production cost, making it difficult to be widely used in domestic clinical practice

Method used

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Examples

Experimental program
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Effect test

Embodiment Construction

[0010] The eukaryotic expression vector pCSRK of the constructed t-PA deletion / point mutant cDNA was transfected into CHO-dhfr by liposome-mediated method - , after the double selection of G418 and DMEM, the stable expression cell line CHO-dhfr was obtained for the first time - pCSRK (Accession No.: CGMCC NO.1094);

[0011] The above t-PA deletion / point mutant cDNA is based on the t-PA V4-E175 deletion mutant cDNA, further carrying out KHRR296-299AAAA point mutation to escape the inhibition of PAI-1.

[0012] And its biological characteristics were identified; in the experiment, the application of serum free medium (serum free medium, SFM) in the production of rPA mutants was explored for the first time, which further proved the role of SFM in the production of medicinal proteins. Broad prospects. The invention lays a good foundation for the clinical development and application of a novel long-acting t-PA that prolongs the half-life, reduces system bleeding tendency, improve...

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Abstract

The invention is a tissue type plasminogen activator mutant rPA(K) gene transfected cell strain, using liposome mediated transformation to transfect the Eukaryotic expression carrier pCSRK of the composed t-PA Cdna deleted / point mutant rPA(K)-rPA gene (KHRR296-299AAAA) (this mutant can escape the inhibition by PAI-1) with CHO-dhfr-, and by double selection of G418 and DMEM, obtaining its stable expression cell strain CHO-dhfr-pCSRK (conservation number: CGMCC No.1094).

Description

Technical field: [0001] The invention relates to the field of cell biology and molecular biology, and its technology belongs to the related experimental methods and technologies of cell biology and molecular biology, especially a kind of tissue-type plasminogen activator deletion / point mutant gene transfection cell strain. Background technique: [0002] Thrombotic complications caused by cardiovascular diseases seriously threaten human life and health, and are one of the main diseases leading to death and disability. There are as many as tens of millions of cardiovascular disease patients in the world every year; the number of thrombotic diseases in my country has also increased significantly in recent years, reaching 3 million per year, and the recurrence rate after treatment is relatively high. The death rate of acute myocardial infarction (AMI) caused by thrombus can be as high as 30%. For many AMI patients, thrombolytic therapy is the method of choice to improve surviv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/57C12N15/58C12N15/85
Inventor 王玉炯李敏扈荣良罗惠霞
Owner NINGXIA UNIVERSITY
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