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Technological process of producing recombinant human histiotype plasminogen activator TNK mutant

A technology of plasminogen and process method, applied in the field of production of recombinant human tissue plasminogen activator mutants, capable of solving the problem of inhibited protease activity and the production method of recombinant human tissue plasminogen activator mutants There are no literature reports and other problems, and the effect of simple equipment, low cost and simple process conditions is achieved

Inactive Publication Date: 2004-01-21
石药集团明复乐药业(广州)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

3. Plasminogen activator inhibitor PAI, t-PA combined with protease activity is inhibited
[0010] There is no literature report on the production method of recombinant human tissue plasminogen activator mutant rhTNK-tPA

Method used

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  • Technological process of producing recombinant human histiotype plasminogen activator TNK mutant
  • Technological process of producing recombinant human histiotype plasminogen activator TNK mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0022] 1 Construction of engineered cell lines

[0023] 1.1 Materials, methods and results

[0024] 11.1 Materials

[0025] 1.1.1.1 Strains

[0026] E. coli XL1-Blue (genotype hsdR17, supE44, recA1, endA1, gyrA46, thi, relA1, lac / F'[proAB+, lac Iq, lacZDM15::Tn10(tetr)]).

[0027] 1.1.1.2 Cell lines

[0028] CHO (dhfr - ), dihydrofolate reductase-deficient Chinese hamster ovary cells.

[0029] 1.1.1.3 Tool enzymes

[0030] Various restriction enzymes, T4 DNA ligase, alkaline phosphatase, and Taq DNA polymerase were purchased from Promega. Vent DNA polymerase was purchased from Biolab Company. DNA molecular weight standards DL2000 (2000, 1000, 750, 500, 250, 100bp) and lamda-DNA / EcoRI (23130, 9416, 6557, 4361, 2322, 2027, 564, 125bp) were purchased from Treasure Bioengineering Dalian Co., Ltd.

[0031] 1.1.1.4 Main reagents

[0032] 1.1.1.4.1 3mM HT (hypoxanthine + thymine) concentrate

[0033] Hypoxanthine (purchased from Sigma Company) 40.83 mg, thymine (Sigma Compa...

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Abstract

The present invention discloses the technological process of producing recombinant human histiotype plasminogen activator TNK mutant. TNK-tPA gene containing said mutant sites is synthesized via full length gene synthesis process and expressed in Chinese hamster ovary cell (CHO-DHFR), and through cloning and screening high-level expressed TNK-tPA engineering cell strain is obtained. By means of sustained culture of the engineering cell in cell culturing bioreactor, collection of the supernatant and serial separation and purification in chromatographic column, rhTNK-tPA product is obtained. The destination gene of the present invention has expression level in cell as high as 18000 IU / 106 cell / d, and the prepared rhTNK-tPA product has single strand rate as high as 80 % and purity up to 95 %. As for industrial scale, the present invention has relatively simple requirement in apparatus, low cost and simple technological requirement.

Description

technical field [0001] The invention relates to a method for producing recombinant human tissue plasminogen activator mutants, in particular to a method for producing recombinant human tissue plasminogen activator TNK mutants (rhTNK-tPA for short) using animal cells process method. Background technique [0002] Tissue plasminogen activator (t-PA) is one of the important components of the coagulation and fibrinolytic system in human blood. It activates the fibrinolytic system by activating plasminogen on the surface of thrombus. Plays an important role in maintaining the balance of coagulation and fibrinolysis. t-PA is synthesized by vascular endothelial cells. The newly synthesized t-PA precursor consists of 562 amino acid residues. During the translation, secretion and maturation of the polypeptide, the N-terminal signal peptide and leader peptide are excised, and the mature t-PA The PA molecule contains 527 amino acid residues, with a molecular weight of 67-72KD. It is c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/09C12P21/02
Inventor 刘龙斌杨琴王敏荣王军志
Owner 石药集团明复乐药业(广州)有限公司
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