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Recombinant glucokinase as thrombolytic medicine

A staphylokinase and thrombolytic technology, which is applied in the field of recombinant staphylokinase, a thrombolytic drug, can solve problems such as being unsuitable for industrialized production and treatment of thrombosis, and achieve the effects of reducing bleeding side effects, strong thrombolytic ability and good specificity

Inactive Publication Date: 2004-04-14
通化康缘玉金药业有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

"Bioengineering" 1994 Volume 12 Issue 2 article "High Yield Production and Purification of Recombinant Staphylokinase for Treating Thrombosis" introduced the production of staphylokinase by transfecting Escherichia coli TG1 cells in a fermenter, and its expression amount accounted for 10% of the total protein of the bacteria. 10%-15%, is not suitable for industrial production of staphylokinase for the treatment of thrombosis

Method used

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  • Recombinant glucokinase as thrombolytic medicine
  • Recombinant glucokinase as thrombolytic medicine
  • Recombinant glucokinase as thrombolytic medicine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1, the source and primary clone of recombinant staphylokinase gene of the present invention, such as figure 1 shown

[0031] Screen positive strains of staphylokinase from the collected 179 strains of Staphylococcus aureus, select strain No. 66 with strong staphylokinase activity, induce the prophage with mitomycin, make it fragmented, and isolate the prophage carrying the staphylokinase genome from the lysate Lysogenize phage α, extract phage DNA, digest with Hind III endonuclease, connect with plasmid pBR322 by shotgun method, and transform Escherichia coli EcoliC 600 The build process is as follows. Screening obtained three transformants carrying exogenous genes (such as figure 2 shown). Its plasmid pSak-1 was digested and electrophoresed, and one of them had the ability to produce staphylokinase (such as image 3 shown). Compared with the standard molecular weight, it shows that it contains about 2.7Kb fragment of staphylokinase gene.

Embodiment 2

[0032] Embodiment 2, the construction of the secondary clone of the recombinant staphylokinase gene of the present invention, as shown in Figure 4:

[0033] On the basis of primary cloning, the connection with the plasmid PUCBM20 as the carrier was digested with EcoR I to obtain the plasmid PUC-E, and the PUC-E plasmid was digested with Hind III. The recovered 4.7Kb fragment was circularized to obtain a 2.1Kb secondary clone.

Embodiment 3

[0034] Embodiment 3, the sequence determination of staphylokinase secondary clone gene fragment:

[0035] A fragment of about 2100 bp was excised with EcoR I for the secondary clone PUC-E, and transferred to MBmp18 phage by the Sanger dideoxy chain termination method. The model in the sequence was AppledBiostens373. Here, a reading frame structure (1028-1517) is found in the sequence of 2001bp, which encodes a protein of 163 amino acids with a molecular weight of 18.476KD, including a signal peptide and a sequence of 136 amino acids of staphylokinase (such as Figure 5a , Figure 5b , Figure 5c shown).

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Abstract

The present invention discloses one gluicokinase gene and its high expression engineering strain, that is, coding Sak gene obtained with DNA of lysogenic conversion phage alpha of Staphylococcus aureus through PCR amplification. The built glucokinase engineering strain has expression amount as high as 25% and is suitable for industrial production. The research of the applicant shows that recombinant glucokinase has specific effect on fibirin, has the features of powerful thrombolytic capacity and less hemorrhagic side effect and is safe and effective.

Description

technical field [0001] The present invention is a thrombus-dissolving drug recombinant staphylokinase, which belongs to the DNA genetic engineering in the field of biomedicine, and relates to a thrombus-dissolving drug recombinant staphylokinase (Sak) gene and its high-expression engineering strain. Proteins have high degradation activity. technical background [0002] Staphylokinase (staphylokinase Sak) is a new type of biotechnology therapeutic drug born in the 1990s. It is an extracellular protein secreted by Staphylococcus aureus. It is similar to urokinase (Uk) and streptokinase (Sk) and has a thrombolytic effect. characteristic. Currently, urokinase, streptokinase, etc. are mainly used in the clinical treatment of thrombosis, which have slow dissolution rate of thrombus, poor thrombolytic effect, and poor specificity to fibrin that constitutes the thrombus skeleton, which can easily lead to abnormalities in the blood system. Fibrinogen is also degraded, thereby activ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/50C12N9/70C12N15/58C12N15/70
Inventor 王家驯
Owner 通化康缘玉金药业有限公司
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