CN1283787C - Cell strain transfacted by deletion multant gene using histo type cellulolytic proenzyme as activator - Google Patents
Cell strain transfacted by deletion multant gene using histo type cellulolytic proenzyme as activator Download PDFInfo
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- CN1283787C CN1283787C CN 200410004736 CN200410004736A CN1283787C CN 1283787 C CN1283787 C CN 1283787C CN 200410004736 CN200410004736 CN 200410004736 CN 200410004736 A CN200410004736 A CN 200410004736A CN 1283787 C CN1283787 C CN 1283787C
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Abstract
The present invention relates to a cell strain transfected by a deletion multant gene using a histiotype cellulolytic proenzyme as an activator. The stable expression cell strain CHO-dhfr<->pCSRA (the storage number is CGMCC No. 1093) is obtained by a liposome mediating method after dual selection of G418 and DMEM on the t-PA containing deletion multant. A series of biology characteristic identification for the cell strain is carried out.
Description
Technical field:
The invention belongs to cytobiology and biology field, its technology relates to cytobiology and molecular biological related experimental methods and technology, particularly a kind of tissue-type plasminogen activator deletion mutant gene transfecting cell.
Background technology:
The thrombus complication serious threat mankind's that cardiovascular disorder causes life is to cause one of dead and invalid principal disease with healthy.The annual cardiovascular patient in the whole world reaches several ten million; The generation number of China's thrombotic diseases also obviously rises in recent years, annual up to 3,000,000, and treatment back recurrence rate is higher, and Acute Myocardial Infarction (the Acutemyocardial infarction that causes by thrombus, AMI) mortality ratio can be up to 30%, for many AMI patients, thromboembolism treatment is the prefered method that improves survival rate and keep left ventricular function, so during seizure of disease, carry out that special effect medicine therapeutic is sanguimotor normally unimpeded to guarantee timely and effectively, very important to the survival rate that improves the patient.
At present clinically official approval use and the thrombolytics tried out can be divided into the three generations, wherein, (the tissue-typeplasminogen activator of tissue-type plasminogen activator as one of s-generation thrombolytics, t-PA) be naturally occurring physiological activator in the fibrinolytic system in the blood, its specifically plasminogen activation change plasmin into, and the latter can be in the thrombus part scleroproein in the thrombus effectively, make revascularization, compare with other thrombolytics, it is little that t-PA has the systemic bleeding of causing tendency, coagulates the weak advantage of trend behind the thrombolysis again; Therefore can bring into play efficient, special thrombolytic effect.But, although t-PA has a lot of benefits clinically, its thromboembolism treatment usually is restricted also, this mainly is because the transformation period of t-PA in blood very lacked (3～5 minutes), making in clinical treatment must heavy dose of intravenous drip for keeping its effective concentration, causes systemic danger of bleeding thereby increased.In addition, during thromboembolism treatment, the use of a large amount of t-PA also makes its price make us being difficult to accepting.Therefore design and development prolong half-life reduce system's bleeding tendency, improve specific activity, simplify route of administration, low-cost novel t-PA ever more important; Based on this, people make great efforts to develop novel third generation thrombolysis reagent.
As the rPA (Reteplase, the V4 of t-PA～E175 deletion mutant) of one of third generation thrombolysis preparation,, to compare just by the authentication of FDA as far back as 1996 with wild-type t-PA, it has many advantages, prolongs relatively as the transformation period; During to patient's medication, mode of injecting but not instiling or the like can be adopted, but, the work of a large amount of purifying and renaturation aspect need be carried out because rPA is colibacillary expression product, thereby increased production cost, made it be difficult to clinically at home be widely used.
And for the eukaryotic cell expression system since its correctly montage process sophisticated mRNA, the probability of raising product correct configuration, and expression product has genetic stability and repeatability, can be secreted in the substratum, purifying technique is simple, and cost is also lower.
Summary of the invention:
The purpose of this invention is to provide a kind of deletion mutant gene transfecting cell strain CHO-dhfr of tissue-type plasminogen activator that obtains with the eukaryotic cell expression method
-PCSRA (preserving number: CGMCC No.1093).
Purpose of the present invention realizes by following scheme:
The deletion mutant gene transfecting cell strain of a kind of tissue-type plasminogen activator, it is the carrier for expression of eukaryon pCSRA transfection CHO-dhfr with t-PA V4～E175 deletion mutant cDNA
-Cell after the double selection of G418 and DMEM, obtains its stable expression cell strain CHO-dhfr first
-PCSRA (preserving number: CGMCC No.1093).
The present invention identifies above-mentioned cell strain biological characteristics.Wherein, the FAPA detected result shows that expression product has good cellulolytic activity; 80 ℃ of dull and stereotyped deactivation experiments show that product has specific activity; Western blotting result shows that product has specific antigenicity; Stability test shows that cell strain has satisfactory stability.In the experiment, also the cell strain characteristic under serum free medium CHO-S-SFM II and the conventional substratum DMEM culture condition is compared, 24h wherein, 48h, during 72h, the expression amount of purpose product, the former all is about 2 times of the latter; SDS-PAGE shows that the former only has a spot of protein band, and the latter then has the amounts of protein band.
Embodiment:
The present invention is the carrier for expression of eukaryon pCSRA transfection CHO-dhfr of the t-PA V4～E175 deletion mutant cDNA that will make up voluntarily with liposome mediated-method
-Cell after the double selection of G418 and DMEM, obtains its stable expression cell strain CHO-dhfr
-PCSRA (preserving number: CGMCC No.1093).And a series of biological characteristicses evaluations have been carried out in the pair cell strain.
Also (serum free medium SFM) is exploring aspect the rPA production application, thereby has proved that further SFM is for producing the proteinic bright prospects of medical to serum free medium first in the experiment.The present invention is development and application prolong half-life clinically, reduces system's bleeding tendency, improves specific activity, and the t-PA that simplifies route of administration and low-cost New-type long-acting has established good basis.
Claims (1)
1. tissue-type plasminogen activator's deletion mutant gene transfecting cell strain is characterized in that it is that preserving number is the cell strain CHO-dhfr-pCSRA of CGMCC No.1093.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410004736 CN1283787C (en) | 2004-03-01 | 2004-03-01 | Cell strain transfacted by deletion multant gene using histo type cellulolytic proenzyme as activator |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410004736 CN1283787C (en) | 2004-03-01 | 2004-03-01 | Cell strain transfacted by deletion multant gene using histo type cellulolytic proenzyme as activator |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1560240A CN1560240A (en) | 2005-01-05 |
| CN1283787C true CN1283787C (en) | 2006-11-08 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN 200410004736 Expired - Fee Related CN1283787C (en) | 2004-03-01 | 2004-03-01 | Cell strain transfacted by deletion multant gene using histo type cellulolytic proenzyme as activator |
Country Status (1)
| Country | Link |
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| CN (1) | CN1283787C (en) |
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2004
- 2004-03-01 CN CN 200410004736 patent/CN1283787C/en not_active Expired - Fee Related
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| CN1560240A (en) | 2005-01-05 |
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