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Perinereis aibuhitensis plasmin, preparation method and uses thereof

A technology of didentate clamworm and fibrinolytic protease, which is applied in the field of biochemistry, can solve the problems of low specificity of dissolving fibrin, large bleeding side effects, large molecular weight, etc., and achieves the effect of simple operation, simple operation and large load

Inactive Publication Date: 2017-07-04
SHANGHAI UNIV OF T C M
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the above-mentioned technical problems in the prior art, the present invention provides a P. bidentate nereis fibrinolytic protease and its preparation method and application. It solves the technical problems in the prior art of low specificity of dissolving fibrin, large bleeding side effects, easy re-blocking, high toxicity, high molecular weight, and high price

Method used

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  • Perinereis aibuhitensis plasmin, preparation method and uses thereof
  • Perinereis aibuhitensis plasmin, preparation method and uses thereof
  • Perinereis aibuhitensis plasmin, preparation method and uses thereof

Examples

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Embodiment 1

[0047]Take 2.5kg dry body of Nereis bidentata, add 10 times the volume of pH 7.4 20mM phosphate buffer to homogenate, place at room temperature for 4 hours for autolysis, centrifuge and use saturated ammonium sulfate 10%-50% graded salting out, collect after centrifugation The active part was reconstituted with buffer, loaded on a Sephadex-G75 chromatography column equilibrated with pH 7.4 20mM phosphate buffer, eluted with pH 7.4 20mM phosphate buffer, and detected the absorbance of fractions at an ultraviolet wavelength of 280nm , after the main fibrinolytic active sites were collected in fractions, they were desalted with a Sephadex-G25 chromatographic column, freeze-dried and concentrated. Reconstitute the sample with pH 7.4 20mM phosphate buffer, load the sample on a DEAE Sepharose Fast Flow chromatography column equilibrated with pH 7.4 20mM phosphate buffer, and elute with a buffer containing 0-0.6M sodium chloride , the main active fraction was collected, desalted and ...

Embodiment 2

[0049] Take 2.5kg dry body of Nereis bidentata, add 10 times the volume of pH7.4 20mM phosphate buffer to homogenate, place it at room temperature for 6 hours for autolysis, centrifuge and use saturated ammonium sulfate 10%-50% graded salting out, and collect after centrifugation The active part was reconstituted with buffer, loaded on a Sephadex-G75 chromatography column equilibrated with pH 7.4 20mM phosphate buffer, eluted with pH 7.4 20mM phosphate buffer, and detected the absorbance of fractions at an ultraviolet wavelength of 280nm , after the main fibrinolytic active sites were collected in sections, they were desalted and concentrated with a 5kDa ultrafiltration centrifuge tube. Reconstitute the sample with pH 7.4 20mM phosphate buffer, load the sample on a DEAE Sepharose Fast Flow chromatography column equilibrated with pH 7.4 20mM phosphate buffer, and elute with a buffer containing 0-0.6M sodium chloride , collect the main active part, use 5kDa ultrafiltration centr...

Embodiment 3

[0051] Take 2.5kg dry body of Nereis bidentata, add 10 times the volume of pH7.4 20mM Tris buffer to homogenate, leave it at room temperature for 6 hours for autolysis, centrifuge and use saturated ammonium sulfate 20%-60% for graded salting out , after centrifugation, the active fraction was collected, redissolved with a buffer, and loaded on a Sephadex-G75 chromatographic column equilibrated with a pH 7.4 20mM Tris buffer, buffered with a pH 7.4 20mM Tris buffer The fractions were eluted with an ultraviolet wavelength of 280nm to detect the absorbance of the fractions, and after the main fibrinolytic active sites were collected in fractions, they were desalted with a Sephadex-G25 chromatographic column, freeze-dried and concentrated. Reconstitute the sample with pH 7.4 20mM tris buffer, load the sample on the DEAESepharose Fast Flow column equilibrated with pH 7.4 20mM tris buffer, and use 0-0.6M chlorine The sodium chloride buffer was eluted, and the main active fraction wa...

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Abstract

The present invention discloses Perinereis aibuhitensis plasmin, which contains amino acid sequences represented by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, has the molecular weight of 33.03 kDa, has the isoelectric point of 5.28, and has the fibrinolytic activity of 10000+ / -1000 U / mg. The invention further provides a preparation method of the Perinereis aibuhitensis plasmin, wherein the preparation method comprises: pre-treating Perinereis aibuhitensis, carrying out gel column chromatography on crude Perinereis aibuhitensis enzyme, and then sequentially carrying out gel filtration, ultra-filtration desalination, ion exchange column chromatography and electrophoresis preparation so as to obtain the Perinereis aibuhitensis plasmin having strong fibrinolytic activity, wherein the obtained Perinereis aibuhitensis plasmin can be used as the thrombotic disease treatment bulk drug. According to the present invention, the method has the simple operation and is controllable, and the Perinereis aibuhitensis plasmin with characteristics of high fibrinolytic activity and high purity can be prepared with the method of the present invention.

Description

technical field [0001] The invention belongs to the field of biochemistry, and relates to a protease, in particular to a didentate clam worm fibrinolytic protease, a preparation method and application thereof. Background technique [0002] Cardiovascular and cerebrovascular diseases are a serious threat to human beings. Thrombotic diseases belong to cardiovascular and cerebrovascular diseases and have become the "number one killer" that seriously endangers human life and health. Controlling the spread of cardiovascular and cerebrovascular diseases has become the top priority of improving people's healthy living standards in the 21st century. Thrombolytic drugs commonly used clinically at home and abroad include urokinase (UK), streptokinase (SK), staphylokinase (staphyLokinase, SaK), tissue type plasminogen activator (tissuetype pLasminogen activator, t-PA) , snake venom defibrase (Diffenzyme, Viper antithrombotic enzyme) and lumbrokinase (LK), etc. t-PA is expensive, UK bl...

Claims

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Application Information

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IPC IPC(8): C12N9/68A61K38/48A61P7/02
Inventor 王新宏梁琨缪潇瑶安叡尤丽莎吴婉莹
Owner SHANGHAI UNIV OF T C M
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