Fibrinolytic enzyme from gloydius intermedius venom and preparation method and application thereof

A technology for plasmin and plasmin activity, which is applied in the field of intermediary pit viper venom plasmin and its preparation and application, can solve the problems of low plasmin, side effects, low plasmin purity, etc. The effect of impurities

Active Publication Date: 2020-01-24
SHAANXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the content of plasmin in Agkistrodon halys venom is very low, the purity of plasmin purified by column chromatography separation technology is not high, and the impurities contained in it will cause side effects

Method used

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  • Fibrinolytic enzyme from gloydius intermedius venom and preparation method and application thereof
  • Fibrinolytic enzyme from gloydius intermedius venom and preparation method and application thereof
  • Fibrinolytic enzyme from gloydius intermedius venom and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0071] The gene of the intermediary Agkistrodon venom fibrinolytic enzyme in this embodiment is Gisp6, which has the nucleotide sequence shown in SEQ ID No. 1 in the sequence listing, and has the amino acid sequence shown in SEQ ID No. 2 in the sequence listing.

[0072] The above-mentioned preparation method of Agkistrodon halys venom fibrinolytic enzyme consists of the following steps:

[0073] (1) The purification method of intermediary Agkistrodon halys venom fibrinolytic enzyme

[0074] 1) Gel filtration chromatography separation

[0075] Dissolve the lyophilized powder of Agkistrodon medidae in 50mM ammonium acetate buffer to prepare a 50mg / mL sample solution, use Sephacry1-200HR as a filler, and use 30-60mM acetic acid containing 0.10-0.25M NaCl and pH 7.3 Ammonium was used as the buffer solution and put on a gel filtration chromatography column. According to the conventional method, gel filtration was performed, and the flow rate was set at 0.45mL / min. A total of 6 pe...

Embodiment 2

[0110] The intermediary Agkistrodon venom fibrinolytic enzyme of this embodiment, its gene is Gisp6, has the nucleotide sequence shown in SEQ ID No.1 in the sequence listing, and has the amino acid sequence shown in SEQ ID No.2 in the sequence listing.

[0111] The preparation method of the intermediate Agkistrodon venom fibrinolytic enzyme of the present embodiment is made up of following steps:

[0112] 1) Gel filtration chromatography separation

[0113] Dissolve the lyophilized powder of Agkistrodon medidae in 50mM ammonium acetate buffer to prepare a 50mg / mL sample solution, use Sephacry1-200HR as the filler, and use 30mM ammonium acetate containing 0.10M NaCl and pH 7.3 as the buffer , put on a gel filtration chromatography column, perform gel filtration according to the conventional method, set the flow rate to 0.45mL / min, a total of 6 peak components appear, and collect the primary component P3 with plasmin activity.

[0114] 2) Anion exchange chromatography separatio...

Embodiment 3

[0120] The intermediary Agkistrodon venom fibrinolytic enzyme of this embodiment, its gene is Gisp6, has the nucleotide sequence shown in SEQ ID No.1 in the sequence listing, and has the amino acid sequence shown in SEQ ID No.2 in the sequence listing.

[0121] The preparation method of the intermediate Agkistrodon venom fibrinolytic enzyme of the present embodiment is made up of following steps:

[0122] 1) Gel filtration chromatography separation

[0123] Dissolve the lyophilized powder of Agkistrodon medidae in 50mM ammonium acetate buffer to prepare a 50mg / mL sample solution, use Sephacry1-200HR as the filler, and use 60mM ammonium acetate containing 0.25M NaCl and pH 7.3 as the buffer , put on a gel filtration chromatography column, perform gel filtration according to the conventional method, set the flow rate to 0.45mL / min, a total of 6 peak components appear, and collect the primary component P3 with plasmin activity.

[0124] 2) Anion-exchange chromatography separatio...

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Abstract

The invention provides a fibrinolytic enzyme from gloydius intermedius venom. The gene of the fibrinolytic enzyme is Gisp6, which has a nucleotide sequence as shown in SEQ ID No.1 of the sequence table and an amino acid sequence as shown in the SEQ ID No.2 of the sequence table. A preparation method of the fibrinolytic enzyme consists of three steps: a purification method of the gloydius intermedius venom fibrinolytic enzyme, purity identification and mass spectrometry analysis, and cloning of the gloydius intermedius venom fibrinolytic enzyme Gisp6 gene. The invention also provides application of the gloydius intermedius venom fibrinolytic enzyme in the establishment of a stable expression cell line in HEK293T cells and application of the fibrinolytic enzyme in thrombolytic drugs. By theadoption of the optimal salt concentration, the non-specific adsorption of components and fillers is reduced. By adopting the optimal pH value, peaks do not overlap, impurities are eliminated, and product purity is improved.

Description

technical field [0001] The invention belongs to the technical field of enzymes, and in particular relates to intermediary Agkistrodon venom fibrinolytic enzyme. Background technique [0002] Thrombus is a blood clot formed by blood components on the damaged surface of vascular endothelial cells and exposed collagen fibers. The main components of thrombus are deposited platelets, insoluble fibrin gel and red blood cells. Thrombus is usually divided into six categories, mixed thrombus includes thrombus head (platelet + a small amount of fibrin), thrombus body (platelet trabeculae + fibrin network + red blood cells) and thrombus tail (fibrin network + red blood cells), transparent thrombus (microthrombus ) is mainly composed of fibrin. In the blood circulation system of the human body, there are two mutually antagonistic and interdependent physiological action systems, namely the coagulation system and the anticoagulation system, namely the fibrinolytic system, referred to as ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64C12N15/57C12N15/85A61K38/48A61P7/02
CPCC12N9/6418C12Y304/24072C12N15/85A61K38/4886A61P7/02
Inventor 杨章民张扭王璐陈毓刘珍珍张腾裴剑竹王喆之
Owner SHAANXI NORMAL UNIV
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