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Prepn process and medicine composition of natural arginine esterase

An arginine esterase and arginine ester technology, applied in the field of biomedicine, can solve the problems of thrombin-like protein structure and property identification, difficulty in improving production efficiency, and intractable drugs, and achieve product purity, quality and ratio. High activity, low production cost, and the effect of eliminating toxic and side effects

Inactive Publication Date: 2007-11-07
SHANGHAI JIAO TONG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since this method uses gel filtration chromatography as one of the preparation methods, it has the disadvantages of low sample loading, low yield, and difficulty in improving production efficiency.
At the same time, because the study did not fully identify the protein structure and properties of the prepared thrombin, it is difficult to be used as a therapeutic drug

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The preparation of the natural arginine esterase of embodiment 1 Agkistrodon halys

[0033] Take by weighing 15g of Agkistrodon acutus venom crude product, dissolve in 160ml sodium phosphate buffer (5mM sodium phosphate, 1mM EDTA, pH6.0), filter to remove insoluble matter, clear sample up to pre-washed with 10 times volume of sodium phosphate buffer (containing 10mM sodium phosphate and 1mM EDTA, pH 6.0) equilibrated with triazoxide immobilized arginine affinity chromatography column (5 × 10cm), sequentially with sodium phosphate buffer (containing 10mM sodium phosphate and 1mM EDTA, pH 6.0) to 280nm light absorption to the baseline, elute with glycine-sodium hydroxide buffer (10mM, pH10.3), collect step by step, and combine components with fibrinogen initial coagulation activity to obtain the natural arginine Crude esterase. Determination of total protein 150mg by folin phenol assay; determination by fibrinogen primary coagulation method, the total activity is about 8...

Embodiment 2

[0040] Embodiment 2 Preparation of natural arginine esterase from Agkistrodon halys

[0041] Weigh 20g of Changbai Mountain Agkistrodon halys viper venom, dissolve it in 200ml sodium phosphate buffer (containing 5mM sodium phosphate and 1mM EDTA, pH 6.0), and load the sample to 10 times the volume of sodium phosphate buffer (containing 10mM sodium phosphate and 1mM EDTA, pH 6.0) equilibrated with triazoxide immobilized arginine affinity chromatography column (2.5 × 20cm), washed successively with sodium phosphate buffer (containing 10mM sodium phosphate and 1mM EDTA, pH 6.0) to 280nm light absorption to Baseline, glycine-sodium hydroxide buffer (10mM, pH 10.6) elutes the bound protein, collects it step by step, and combines the components with fibrinogen primary coagulation activity to obtain the crude product of natural arginine esterase. Determination of total protein 130mg by Folin phenol assay; Determination by fibrinogen primary coagulation method, the total activity is 2...

Embodiment 3

[0048] Embodiment 3 measures natural arginine esterase molecular mass

[0049] Take the refined product of natural arginine esterase and mix it with sinapinic acid matrix, put about 1 μl on the target, let it dry naturally, put it into a mass spectrometer (manufactured by Brukerg, model AutoFlex MALDI-TOF-TOF-MS), N 2 Laser source (wavelength 337nm); positive ion linear detection (flight tube length 1.22M, acceleration voltage 20KV) to obtain MALDI-TOP MS spectrum, there are mass peaks at m / z 17503, 34069, 68138, which are half molecular masses respectively , molecular mass and double molecular mass, so the molecular mass of natural arginine esterase is 34.07±0.20kDa.

[0050] Determination of the sugar content of natural arginine esterase molecules

[0051] Take the refined natural arginine esterase solution (52 μg / ml) by desalting and freeze-drying, add 20 μl trifluoromethanesulfonic acid (TFMS), react overnight at -70°C, then add 1200 μl of pre-cooled acetonitrile at -20°C...

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Abstract

The present invention is one kind of natural arginine esterase and its preparation process and medicine composition, and belongs to the field of biomedicine technology. The process of preparing natural arginine esterase includes the following steps: adsorbing natural arginine esterase from coarse snake venom material with affinity column mounted with triazozine fixed L-arginine separating material, eluting with alkaline buffering and collecting the eluted component to obtain coarse arginine esterase product, and final ion exchange chromatography to refine and to obtain refined natural arginine esterase product. The medicine composition of natural arginine esterase contains natural arginine esterase in 0.000083-0.129 wt% and medicine carrier and supplementary material in 99.871-99.999917 %. The natural arginine esterase and its medicine composition may be used in preventing and treating various kinds of thrombus and embolic diseases.

Description

technical field [0001] The invention relates to an enzyme preparation method and its pharmaceutical composition, in particular to a natural arginine esterase preparation method and its pharmaceutical composition. It belongs to the technical field of biomedicine. Background technique [0002] Snake venom-like thrombin is a general term for a class of enzymes in snake venom that are similar in properties to plasma thrombin, and usually have arginine esterase activity, also known as arginine esterase. It can enzymatically decompose fibrinogen in the blood, but does not activate various coagulation factors in the body. Therefore, plasma or fibrin solution can be coagulated in vitro, while the fibrin clot generated in vivo has a loose structure and is easily removed by the fibrinolytic system, so that the concentration of fibrinogen is significantly reduced, showing anticoagulant effect. This characteristic makes arginine esterase an important source for the development of thro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18A61K38/46
Inventor 李荣秀辛瑜王霆
Owner SHANGHAI JIAO TONG UNIV
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