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 combination, pharmaceutical formula, medical preparations of non-active ingredients, etc., can solve the problem of biological activity reduction, achieve half-life improvement, biological activity retention, and great application potential Effect

Inactive Publication Date: 2012-05-16
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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1. Preparation of polyethylene glycol (20000)-staphylokinase conjugate

[0019] Recombinant human staphylokinase was dissolved in 50mM pH5.0 sodium acetate buffer to prepare a 1mg / mL solution, and the ratio of staphylokinase: PEG aldehyde (molecular weight: 20000): reducing agent (sodium cyanoborohydride) to 1:4: The reaction was carried out at a molar ratio of 40. The reaction was carried out at 4°C for 12 hours, and 1M glycine was added to terminate the reaction. The modified mixture was purified with Superdex 200, and the PEG-staphylokinase conjugate was collected. The eluent was 0.05 mol / L phosphoric acid Buffer (containing 0.15M NaCL), pH 7.0.

Embodiment 2

[0020] Example 2. Preparation of polyethylene glycol (5000)-staphylokinase conjugate

[0021] Recombinant human staphylokinase was dissolved in 50mM pH5.0 sodium acetate buffer to prepare a 1mg / mL solution, according to staphylokinase: PEG aldehyde (molecular weight 5000): reducing agent (sodium cyanoborohydride) 1: 2: The reaction was carried out at a molar ratio of 20 and reacted at 4°C for 12 hours. The reaction was terminated by adding 1M glycine. The modified mixture was purified with Superdex 200, and the PEG-staphylokinase conjugate was collected. The eluent was 0.05 mol / L phosphoric acid Buffer (containing 0.15M NaCL), pH 7.0.

Embodiment 3

[0022] Example 3. Preparation of polyethylene glycol (40000)-staphylokinase conjugate

[0023] Recombinant human staphylokinase was dissolved in 50mM pH5.0 sodium acetate buffer to prepare a 1mg / mL solution, and the ratio of staphylokinase: PEG aldehyde (molecular weight 40,000): reducing agent (sodium cyanoborohydride) to 1:6: The reaction was carried out at a molar ratio of 60. The reaction was carried out at 4°C for 12 hours, and 1M glycine was added to terminate the reaction. The modified mixture was purified with Superdex 200, and the PEG-staphylokinase conjugate was collected. The eluent was 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 greatapplication potential can be realized in the preparation of cardiovascular and cerebrovascular disease treatment medicine.

Description

Technical field [0001] The present invention relates to the field of biopharmaceuticals. Specifically, the present invention relates to a polyethylene glycol-staphylokinase conjugate and a preparation method and application thereof. Background technique [0002] Staphylokinase is derived from Staphylococcus aureus and is composed of a single chain of 136 amino acids without disulfide bonds, with a molecular weight of approximately 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 platelet-rich arterial thrombosis. Thrombolytic ability. Since staphylokinase has the above-mentioned excellent fibrinolytic properties, it has become the focus of research and development. At present, the staphylokinase gene has been cloned into Escherichia coli, Bacillus subtilis, ...

Claims

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Application Information

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Patent Type & Authority Applications(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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