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Fibrilolytic enzyme as well as preparation method and application thereof

A technology for fibrinolytic and thrombolytic drugs, applied in the field of protease preparations, can solve the problems of unclear effect in vivo, complicated separation and purification methods, etc., and achieve the effects of good in vitro anticoagulation effect, suitable for mass production, and high enzyme activity

Active Publication Date: 2021-06-25
BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its in vivo effect is not clear, and the separation and purification methods are relatively complicated

Method used

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  • Fibrilolytic enzyme as well as preparation method and application thereof
  • Fibrilolytic enzyme as well as preparation method and application thereof
  • Fibrilolytic enzyme as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1 A kind of preparation method of fibrinase

[0051] 1. Carrier construction

[0052] (1) PCR amplification of SEQ ID NO.2: the amplification forward primer sequence is SEQ ID NO.3, and the reverse sequence is SEQ ID NO.4. The template is the cDNA obtained by reverse transcription after extracting the RNA of Cordyceps militaris. Among them, the Cordyceps militaris preservation number is CGMCC 3.14242; the RNA extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., the article number is DP430; the reverse transcription kit was purchased from Takara, the article number is RR036A. The amplification program is: 95°C, 30s; 94°C, 30s, 55°C, 30s, 72°C, 90s, 35cycles; 72°C, 5min.

[0053](2) Ligation transformation: The PCR product was ligated with the pEASY-Blunt E2 vector. The pEASY-Blunt E2 vector was purchased from Beijing Quanshijin Biotechnology Co., Ltd., the product number was CE211, the reaction temperature was 25°C, and the r...

Embodiment 2

[0060] Embodiment 2 A kind of thrombolytic experiment of fibrinase

[0061] In this embodiment, the in vitro thrombolytic activity of the fibrinolytic enzyme obtained in Example 1 is measured, and the specific methods and steps are as follows:

[0062] Heart blood was collected from healthy SD rats (purchased from Beijing Weitong Lihua Co., Ltd.), divided into 0.6 mL tubes, and the empty tubes of centrifuge tubes were recorded as m1. After the blood was left to coagulate naturally, centrifuge at 3000rpm for 10min to remove the supernatant serum, blot the liquid with filter paper, weigh the mass of the remaining blood clot and the centrifuge tube, and record it as m2.

[0063] Experiments were carried out according to the following groups, with 3 parallels in each group:

[0064] (1) 200 μL of 1×PBS buffer;

[0065] (2) 200 μL of 0.15 g / L urokinase;

[0066] (3) 200 μL of 0.15 g / L streptokinase;

[0067] (4) 0.15g / L fibrinolytic enzyme 200μL;

[0068] (5) 200 μL of 0.45 g / ...

Embodiment 3

[0073] Embodiment 3 A kind of thrombolytic experiment of fibrinase

[0074] Determination of in vivo thrombolytic activity of recombinant enzyme (effect on rat tail vein thrombosis):

[0075] The rat tail vein thrombosis model was used to study the thrombolytic activity of the fibrinolytic enzyme obtained in Example 1 in rats. Select 1.2 mg / kg concentration of k-carrageenan to inject into the tail vein of rats, observe the formation of tail thrombus after 24 hours, and measure the length of the formed wine red thrombus.

[0076] Forty-two Wistar male rats (purchased from Beijing Weitong Lihua Co., Ltd.) were injected with equal volumes in the following groups:

[0077] A. Inject 1×PBS;

[0078] B. Injection of 200 μg / kg fibrinolytic enzyme;

[0079] C. Injection of 600 μg / kg fibrinolytic enzyme;

[0080] D. Injection of 1800 μg / kg fibrinolytic enzyme;

[0081] E. Injection of 3000 μg / kg fibrinolytic enzyme;

[0082] F. Inject 600 μg / kg urokinase;

[0083] G. Inject 600 ...

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Abstract

The invention provides a fibrinolytic enzyme, which is characterized in that the amino acid sequence of the fibrinolytic enzyme is SEQ ID NO. 1, and the nucleotide coding sequence of the fibrinolytic enzyme is SEQ ID NO. 2. The fibrinolytic enzyme provided by the invention is simple in preparation method, the obtained enzyme activity is relatively high, the in-vitro thrombolysis rate can reach 80% or above, the in-vivo thrombolysis capability is better than that of commonly used medicines such as urokinase and streptokinase, and meanwhile, the fibrinolytic enzyme also has a very good in-vitro anticoagulation effect and a very good clinical application prospect.

Description

technical field [0001] The invention belongs to the field of protease preparations, and in particular relates to a fibrinolytic enzyme and its preparation method and application. Background technique [0002] Thrombus is a small block formed by blood flow on the surface of the peeled or repaired part of the inner surface of the cardiovascular system. Intravascular thrombosis is a major cause of cardiovascular and cerebrovascular diseases. In variable flow dependent patterns, thrombi consist of insoluble fibrin, deposited platelets, accumulated leukocytes, and trapped erythrocytes. Cardiovascular intima injury, changes in blood flow, and changes in blood properties may all cause thrombus formation. [0003] At present, the treatment of thrombosis mainly relies on antithrombotic drugs, which can be divided into three categories: anticoagulant drugs, antiplatelet aggregation drugs and thrombolytic drugs, among which thrombolytic drugs can be used to treat acute thromboembolic ...

Claims

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Application Information

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IPC IPC(8): C12N9/58C12N15/57C12N15/85C12N15/70A61K38/48A61P7/02
CPCA61K38/00A61P7/02C12N9/58C12N15/70C12N15/85C12Y304/21007
Inventor 刘英丽王静
Owner BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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