Polyethyleneglycol-staphylokinase conjugate as well as preparing method and application thereof

A technology of polyethylene glycol and staphylokinase coupling, which is applied in the direction of drug combinations, pharmaceutical formulas, medical preparations of non-active ingredients, etc., can solve the problem of biological activity reduction, achieve improved half-life, huge application potential, and retain biological activity Effect

Inactive Publication Date: 2013-10-16
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention is completed in order to solve the problem that PEG-modified staphylokinase in the prior art causes its biological activity to decrease

Method used

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  • Polyethyleneglycol-staphylokinase conjugate as well as preparing method and application thereof
  • Polyethyleneglycol-staphylokinase conjugate as well as preparing method and application thereof
  • Polyethyleneglycol-staphylokinase conjugate as well as preparing method and application thereof

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Embodiment 1

[0018] Embodiment 1, the preparation of polyethylene glycol (20000)-staphylokinase conjugate

[0019] Recombinant human staphylokinase was dissolved with 50mM sodium acetate buffer at pH5.0, and prepared into a solution of 1 mg / mL, according to the ratio of staphylokinase: PEG aldehyde (molecular weight 20000): reducing agent (sodium cyanoborohydride) 1:4: React at a molar ratio of 40, react at 4°C for 12 hours, add 1M glycine to terminate the reaction. Use Superdex 200 to purify the obtained modification mixture, collect the PEG-staphylokinase conjugate, and the eluent is 0.05mol / L phosphoric acid Buffer (containing 0.15M NaCl), pH 7.0.

Embodiment 2

[0020] Embodiment 2, preparation of polyethylene glycol (5000)-staphylokinase conjugate

[0021] Recombinant human staphylokinase was dissolved with 50mM sodium acetate buffer at pH5.0, and prepared into a solution of 1 mg / mL, according to the ratio of staphylokinase: PEG aldehyde (molecular weight 5000): reducing agent (sodium cyanoborohydride) 1:2: React at a molar ratio of 20, react at 4°C for 12 hours, and add 1M glycine to terminate the reaction. Use Superdex 200 to purify the obtained modification mixture, collect PEG-staphylokinase conjugates, and eluate with 0.05mol / L phosphoric acid Buffer (containing 0.15M NaCl), pH 7.0.

Embodiment 3

[0022] Embodiment 3, preparation of polyethylene glycol (40000)-staphylokinase conjugate

[0023] Dissolve the recombinant human staphylokinase with 50mM sodium acetate buffer solution at pH 5.0, and prepare a solution of 1 mg / mL, according to the ratio of staphylokinase: PEG aldehyde (molecular weight 40000): reducing agent (sodium cyanoborohydride) 1:6: React at a molar ratio of 60, react at 4°C for 12 hours, add 1M glycine to terminate the reaction. Use Superdex 200 to purify the obtained modification mixture, collect the PEG-staphylokinase conjugate, and the eluent is 0.05mol / L phosphoric acid Buffer (containing 0.15M NaCl), pH 7.0.

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Abstract

The invention relates to the field of biological pharmacy, in particular to a polyethyleneglycol-staphylokinase conjugate as well as a preparing method and application thereof. The conjugate is characterized in that the N end of staphylokinase is connected with a polyethyleneglycol covalence conjugate, wherein the polyethyleneglycol covalence is connected to the N end of the staphylokinase through polyethyleneglycol modifying agents, the polyethyleneglycol modifying agents optimally adopt methoxy polyethyleneglycol aldehyde modifying agents, the molecular weight of the polyethyleneglycol modifying agents is optimally 1000 to 40000, and the polyethyleneglycol modifying agents comprise line type and branching type structures. The polyethyleneglycol-staphylokinase conjugate provided by the invention has the advantages that the in-vivo half-life period can be greatly prolonged, simultaneously, the biological activity of the original protein can also be completely maintained, and the great application potential can be realized in the preparation of cardiovascular and cerebrovascular disease treatment medicine.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular, the invention relates to a polyethylene glycol-staphylokinase conjugate and its preparation method and application. Background technique [0002] Staphylokinase is derived from Staphylococcus aureus. It consists of 136 amino acids and consists of a single chain without disulfide bonds, with a molecular weight of about 15KD (Eur. J. Biochem. 1985, 149: 557-561). Compared with other thrombolytic agents, staphylokinase has good thrombolytic specificity, does not cause the reduction of fibrinogen and α-antiplasmin levels, is not easy to induce systemic hemolysis, and has a strong effect on arterial thrombus rich in platelets. thrombolytic ability. Because staphylokinase has the above-mentioned excellent fibrinolytic properties, it has become the focus of people's research and development. At present, the staphylokinase gene has been cloned and expressed in Escherichia coli, Bacillus...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K47/48A61K38/16A61P7/02
Inventor 苏志国马光辉王俊刘永东胡涛
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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