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Protease, preparation method of same, as well as application and pharmaceutical formulation thereof

A protease and dosage form technology, applied in the fields of medicine, insects, and enzymology, can solve the problems of short half-life, strong bleeding side effects, and difficult to control the dosage of drugs.

Inactive Publication Date: 2011-09-14
辽宁省农业科学院大连生物技术研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing thrombolytic drugs still have some disadvantages: ① Lack of specificity for thrombolysis; ② Strong bleeding side effects; ③ Short half-life; ④ Difficult to control the dosage;
In Tussah mori, there are no research reports on the separation and purification of this protease, the analysis and identification of enzyme activity, and its role in thrombolysis.

Method used

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  • Protease, preparation method of same, as well as application and pharmaceutical formulation thereof
  • Protease, preparation method of same, as well as application and pharmaceutical formulation thereof
  • Protease, preparation method of same, as well as application and pharmaceutical formulation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment one: the separation and preparation of protease

[0041] Tussah silkworm (Antheraea pernyi) belongs to Lepidoptera (Lepidoptera) Saturniidae, is a kind of economic insect mainly raised in the wild in northern my country. The experimental material used in the present invention is the live tussah silkworm chrysalis sold in the market. Take tussah silkworm chrysalis, incubate at 25°C until the moth emerges, and collect the exudate from the mandibular gland. Take the spit out, put it in a centrifuge tube, adjust the pH to 6.0 with buffer solution 0.02mol / L HAC-NaAC (pH6.0), centrifuge at 12000r / min for 10min, and take the supernatant for later use (if the sample is less than 2ml, use buffer solution 0.02 mol / LHAC-NaAC, pH6.0 make up to 2ml). The cation exchange column (HiTrap TM SP-FF), and then equilibrate with Binding Buffer (0.02mol / L HAC-NaAC, pH 6.0) of 5 times the column volume at a flow rate of 2ml / min. Take 2ml of the sample for loading, and then use ...

Embodiment 2

[0042] Example 2: Analysis of plasmin activity of protease and determination of enzymatic properties

[0043] 1. Degradation of fibrinogen and determination of plasmin activity

[0044] (1) Degradation of fibrinogen

[0045] Take 0.2% human fibrinogen (0.1mol / L Tris-HCL, pH 8.0) solution and 10 microliters of purified protease, mix and incubate at 37°C for 0, 0.5, 1, 2, 3, 4, 8, At 16 and 24 hours, denaturant (10M urea, 4% SDS, 4% 2-mercaptoethanol) was added to stop the reaction, and SDS-PAGE was used for determination. Such as figure 2 , showed that tussah protease could effectively hydrolyze the Aα-chain and Bβ-chain of fibrinogen, but the hydrolysis activity on the γ-chain was weak. The results calculated by Bandscan software are as follows: image 3 As shown in .4, the tussah protease firstly hydrolyzes the Aα-chain of fibrinogen, and can hydrolyze more than 90% within 2 hours; it takes 4 hours to reach 90% for the hydrolysis of the Bβ-chain, and it takes 4 hours for...

Embodiment 3

[0058] Example 3: Analysis of Thrombolytic Effect of Protease in Vitro

[0059] 1. Detection of thrombolytic effect

[0060] Kunming rats were anesthetized with ether, and blood was collected by cardiac puncture. After the blood coagulates, it is cut into uniform small pieces and weighed. The blood clot was added to 0.5 mL tussah protease (0.02 mmol / L NaAC-HAC) solution, and 0.02 mmol / L NaAC-HAC was used as a control, and placed in a constant temperature water bath at 37°C. Every 10 minutes, slightly oscillate, observe, record and take pictures regularly. As a result, it was found that the solution in the upper part of the control was clearer, and the thrombus was not obviously dissolved. The solution contained a very small amount of red blood cells. In the tussah protease group, the solution in the upper part was turbid, and the thrombus was dissolved obviously. The solution contained a large amount of cells released from the thrombus. See the appendix Figure 5 .

[0061...

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Abstract

The invention relates to a protease, a preparation method of same, as well as application and pharmaceutical formulation thereof. A protease is separated and extracted from a spitting liquid secreted before the emergence of an insect with capabilities of spinning and cocooning. The protease with fibrinolysis activity has the characteristics of high bioactivity, excellent stability and the like. The protein has strong arginine esterase activity and can degrade Aa Bb gamma chain in fibrinogen so as to have degradation function to the fibrin and the fibrinogen. As the protease has functions of degrading thrombus of animal and human beings and has the characteristics of not damaging the released blood cells and the like, the protease becomes a new thrombolysis type of medicine for treating diseases related with thrombus.

Description

technical field [0001] The present invention relates to the fields of insects, enzymology, medicine, etc., and relates to the extraction method, thrombolytic effect, gene sequence and encoded amino acid sequence of a protease with plasmin activity secreted from tussah silkworm chrysalis when it emerges, and clinically Application in the treatment of human thrombotic diseases, etc. Background technique [0002] Thromboembolic disease has become the leading cause of human death in developed countries. With the continuous improvement of people's living standards in our country, the incidence of cardiovascular and cerebrovascular diseases is increasing year by year. According to incomplete statistics, about 2 million people die of cardiovascular and cerebrovascular diseases every year. [0003] At present, there are three main treatment methods for thromboembolic diseases: first, surgical removal of thrombus or removal of blood vessels at the embolized site and bridging or repl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/64A61K38/48A61K9/14A61P7/02
Inventor 李树英李文利范琦耿鹏李亚洁赵振军张波
Owner 辽宁省农业科学院大连生物技术研究所
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