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Novel bifunctional molecule of RGD staphylokinase, its preparation process and application

A bifunctional molecule, staphylokinase technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, medical preparations containing active ingredients, etc., can solve the problems of decreased activity and insolubility of RGD-SAK

Inactive Publication Date: 2008-07-09
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to overcome the above-mentioned shortcomings of RGD-SAK activity decline and insoluble expression, the present invention provides a novel RGD-staphylokinase bifunctional molecule with dual activities of thrombolysis and anticoagulation

Method used

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  • Novel bifunctional molecule of RGD staphylokinase, its preparation process and application
  • Novel bifunctional molecule of RGD staphylokinase, its preparation process and application
  • Novel bifunctional molecule of RGD staphylokinase, its preparation process and application

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1 Staphylokinase mutant gene construction, identification and expression of expression plasmid

[0037] 1. Materials and Methods

[0038] 1.1 Materials

[0039]Escherichia coli DH5α, pBV220, recombinant staphylokinase (rSAK) plasmids and strains were prepared by our laboratory (the method of producing staphylokinase by genetic engineering technology, Chinese Invention Patent No. 98102132), the staphylokinase standard was purchased from China Institute for the Control of Biological Products, Taq DNA Polymerase was purchased from Dalian Bao Biological Company, T4 DNA ligase, plasmid extraction kit, restriction endonuclease EcoR I, BamH I were purchased from Promega Company, DNA purification kit was purchased from Dingguo Biotechnology Company, protein purification system It is a product of Pharmacia Company, and the platelet aggregation and hemagglutination tester is a product of Beijing Shidi Scientific Instrument Company. All other reagents were of domestic...

Embodiment 2

[0063] The activity analysis of embodiment two RGD staphylokinase mutants

[0064] 1. Material method

[0065] 1.1 Materials: The preparation and purification of wild-type staphylokinase is the same as the preparation method of the staphylokinase mutant in Example 1. For other materials, see Example 1.

[0066] 1.2 Method:

[0067] 1.2.1 Determination of thrombolytic activity of staphylokinase mutants: with wild-type staphylokinase as a control, the fibrinolytic circle method was used, referring to the 2001 edition of the Chinese Biological Products Testing Regulations, that is, in the presence of fibrinogen, human plasmin and blood coagulation Make holes on the gel plate of the enzyme, dilute the mSAK protein and add to each well, add 10 μl of sample to each well, place it in a humid plate in a 37°C incubator overnight, measure the diameter of the dissolution circle, and use a serially diluted staphylokinase standard at the same time A standard curve was made, and the throm...

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Abstract

The present invention discloses one new type of double function RGD glucokinase molecule and its preparation process and use. The double function RGD glucokinase molecule is glucokinase or its derivative with the amino acids in places 35, 36 and 37 substituted successively into arginine R, glycine G and aspartic acid D. The preparation process of the mutant includes the steps of: site-directed mutation of glucokinase gene, connection to the expression vector, high efficiency expression in colibacillus, separation and purification, etc. The glucokinase mutant of the present invention may be used as thrombolytic medicine and has both thrombolytic and anticoagulant activity.

Description

technical field [0001] The present invention relates to a bifunctional molecule of staphylokinase, its preparation method and application, specifically, the present invention relates to a novel bifunctional molecule of staphylokinase with an RGD sequence, its preparation method and its application in the preparation of anticoagulant and thrombolytic drugs on the application. Background technique [0002] Staphylokinase (Staphylokinase, SAK) is a protein secreted by lysogenic phage in Staphylococcus aureus. Its mature protein consists of 136 amino acids and has a relative molecular weight of 15,000. SAK is an activator of plasminogen (Plg), which forms a 1:1 complex with Plg in plasma, and the complex is activated by trace plasmin (Plasmin, Plm) on the surface of blood clots to SAK-Plm , SAK-Plm is a high-efficiency plasminogen activator, which specifically cuts Plg to form active Plm, dissolves thrombus, and has strong specificity for thrombus dissolution. [0003] As a ne...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00C12N9/70C12N9/12C12N15/52C12N15/55C12N15/54C12N15/11C12N15/70A61K38/43
Inventor 姜永强郑玉玲宁保安马茹
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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