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Thrombolytic drug based on cobra venom piii type metalloprotease and its application

A metalloprotease, cobra venom technology, applied in the direction of peptide/protein components, medical preparations with inactive ingredients, medical preparations containing active ingredients, etc.

Active Publication Date: 2019-08-13
THE KEY LAB OF CHEM FOR NATURAL PROD OF GUIZHOU PROVINCE & CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

SVMP isolated and prepared from Viperidae and Rattlesnake snake venom cannot be used in the preparation of related drugs due to its inherent hemorrhagic toxicity

Method used

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  • Thrombolytic drug based on cobra venom piii type metalloprotease and its application
  • Thrombolytic drug based on cobra venom piii type metalloprotease and its application
  • Thrombolytic drug based on cobra venom piii type metalloprotease and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The target protein is obtained by separating and purifying from the snake venom of Chinese cobra (Zhoushan species cobra) (classification name: Naja atra). The target protein is a single-chain glycoprotein, and its structural analysis shows that it has three structural domains: a metalloprotease domain, a disintegrin-like domain, and a cysteine-rich domain, belonging to the PIII type snake venom metalloprotease ( figure 1 ).

[0034] Table 1 The physical and chemical indicators of the target protein deduced from the primary structure

[0035]

[0036] As shown in Table 1: the target protein has the activity of hydrolyzing the alpha chain of fibrinogen, which can be completely inhibited by metal chelating agents EDTA, EGTA, 1,10-phenanthroline and reducing agent DTT. The actual molecular weights of the target proteins Atrase A and Atrase B are about 49.3kDa and 49.4kDa respectively, and their sequences are: AtraseA: SEQIDNo.1; AtraseB: SEQIDNo.2; however, the apparen...

Embodiment 2

[0052] 1. The role of cobra venom type PIII metalloproteinases in inducing endothelial cell fibrinolysis (taking Atrase A as an example)

[0053] 1.1 Materials and reagents

[0054] Human microvascular endothelial cells (HMEC); RPMI-1640 medium was purchased from Gibco; fetal bovine serum (FBS) was a product of Tianjin Haoyang Biological Products Technology Co., Ltd.; t-PA (tissue plasminogen activator ), PAI-1 (plasminogen activator inhibitor) content detection ELISA kit and activity assay kit were purchased from Assaypro Company of the United States; normal human serum (normal human serum, NHS) was prepared from the blood donation of several healthy volunteers, and frozen Stored at -80°C, tested for normal complement activity, ready for use; inactivated normal human serum (INHS) was obtained by incubating NHS at 56°C for 30 minutes; the separation and purification of Atrase A was the same as before; the rest of the reagents were in compliance with Analytical purity required...

Embodiment 3

[0077] 1. The pharmacodynamic effect of cobra venom type PIII metalloprotease on thrombolysis in vivo (taking Atrase A as an example)

[0078] 1.1 Experimental materials

[0079] The preparation of the thrombolytic drug Atrase A was the same as before, and the purity of the SDS-PAGE test was a single electrophoresis band, and the purified samples were subpackaged and stored at -80°C; aspirin was purchased from Sigma Company in the United States; urokinase (UK) was Livzon The product of Livzon Pharmaceutical Factory of the Group; the recombinant human tissue plasminogen activator alteplase (rt-PA) is a product of Boehringer Ingelheim, Germany; other reagents are all reagents or drugs that meet the experimental requirements.

[0080] SPF-grade male SD rats were purchased from Chongqing Tengxin Experimental Animal Co., Ltd., weighing 210-240 g, with certificate number SCXK (Yu) 2007-0005. Animal experiments and animal welfare complied with relevant animal experiment management r...

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Abstract

The invention a thrombolytic drug based on cobra venom PIIII type metalloproteinase which is obtained by separating and purifying venom of Chinese cobra or Naja cobra with a class name of Naja atra. The metalloproteinase is singe strand glycoprotein, structural analysis shows that the metalloproteinase has three structural domains, namely a metalloproteinase structural domain, a disintegrin structural domain and a cysteine-rich structural domain and belongs to PIIII type metalloproteinase with names of Atrase A and Atrase B. A sequence of the Atrase A is SEQ ID No.1, and a sequence of the Atrase B is SEQ ID No.2. Target protein and the thrombolytic drug can induce endogenous fibrinolytic enzyme activator tPA release so as to play a thrombolytic role, and the thrombolytic drug can be used for thrombolytic treatment of thrombotic diseases.

Description

technical field [0001] The invention relates to the field of application of cobra venom type PIII metalloprotease, in particular to a thrombolytic drug based on cobra venom type PIII metalloprotease and its application. Background technique [0002] Cardiovascular and cerebrovascular diseases are high-incidence and critical diseases in modern society, and the mortality rate remains high, which seriously threatens human life and health. Among them, the hazards of thrombotic diseases are the most serious. Clinically, acute myocardial infarction, ischemic stroke, and acute pulmonary embolism are the main manifestations, and their morbidity, disability, and fatality rates are high. In the past 30 years, thrombolytic therapy has been widely used in the treatment of various vascular thrombotic diseases, and has achieved remarkable results. It has become an effective means for the treatment of thrombotic diseases. my country is a country with a large population. With the developme...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/64A61K38/48A61K9/19A61K47/18A61P7/02
CPCA61K9/0019A61K9/19A61K38/00A61K47/183C12N9/6418
Inventor 孙黔云王彩娥李红玲李亚男
Owner THE KEY LAB OF CHEM FOR NATURAL PROD OF GUIZHOU PROVINCE & CHINESE ACADEMY OF SCI
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