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Armadillidiam vulgare plasmin and application thereof

A technology of fibrinolytic enzymes and fibrinolytic enzymes, applied in fibrinolytic enzymes and its application fields, can solve the problems that cannot be used to prevent thrombosis and poor anticoagulant activity, and achieve the reduction of the risk of tissue hemorrhage, strong thrombolysis and anticoagulation Mild effect of coagulation and thrombolysis

Active Publication Date: 2015-04-29
NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these thrombolytic drugs have greatly reduced the mortality and disability rates of thrombotic diseases, they still have many disadvantages. For example, although urokinase has very good thrombolytic activity, its anticoagulant activity is poor, so urokinase It can only be used for thrombolysis, not for the prevention of thrombosis
Few drugs currently on the market have both thrombolytic and anticoagulant effects

Method used

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  • Armadillidiam vulgare plasmin and application thereof
  • Armadillidiam vulgare plasmin and application thereof
  • Armadillidiam vulgare plasmin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: Preparation of murine fibrinolytic enzyme

[0041] Murine fibrinolytic enzyme of the present invention adopts following method to prepare:

[0042] (1) Wet ultrafine pulverization extraction

[0043] Weigh a certain amount of dried muffins, add 8 times the mass of muffins, soak in distilled water at 25°C for 1 h, put them into an ultrafine pulverizer and pulverize them at 25°C for 10 min. The obtained slurry was heated at 5000 rpm at 4 °C -1 Centrifuge for 10 min and take the supernatant to obtain the extract.

[0044](2) Use hollow fiber membrane to extract protein components with a molecular weight of 4kDa~80kDa

[0045] Pass the extract obtained in step (1) through a PVDF (polyvinylidene fluoride) hollow fiber membrane with a molecular weight cut-off of 80kDa, the operating pressure is 21.34 kPa ~ 21.56kPa, the flow rate is 8ml / min, and the permeate is taken. The permeate is passed through a PES (polyethersulfone) hollow fiber membrane with a molecu...

Embodiment 2

[0053] Example 2. Purification results of murine plasmin PSLTro01

[0054] Adopt the result of method purification in embodiment 1 as Figure 1-4 shown. The separation range of the Sephadex G-100 gel filtration column is 2 kDa-120 kDa, and the chromatogram is as follows figure 1 As shown, a large elution peak and a longer shoulder peak appeared during the elution process, and the protein with thrombolytic activity flowed out of the column before other components, suggesting that the molecular weight of the protein with thrombolytic activity may be greater than 10 kDa. figure 2 The chromatogram of the Sephacryl S-200 HR gel filtration column is shown, and there are two main parallel peaks, and the protein with thrombolytic activity is mainly concentrated in the front part of the second eluting peak. During the elution process of HiTrap Capto Q ion-exchange chromatography column, there are 5 different protein elution peaks, breakthrough peak, low salt elution (the NaCl conce...

Embodiment 3

[0063] Example 3 Determination of the purity, relative molecular mass and N-terminal amino acid sequence of plasminase PSLTro01

[0064] 1. Purity test

[0065] (1) Purity detection by non-reducing SDS-PAGE vertical electrophoresis

[0066] The concentration of the separating gel in the non-reducing SDS-PAGE vertical electrophoresis was 12%, and the concentration of the stacking gel was 3.9%. Take 10 µL of the plasminase PSLTro01 prepared in Example 1 and 2 µL of non-reducing loading buffer (purchased from: Beyond Biotechnology Research Institute), mix thoroughly, and boil at 100°C for 5 min. Electrophoresis was started at a constant voltage of 80 V. After the front of the bromophenol blue band entered the separation gel, the voltage was adjusted to 120 V until the bromophenol blue band reached the bottom of the separation gel. After electrophoresis, silver staining was performed quickly. Such as Figure 5 As shown, only a single electrophoresis band was displayed in the no...

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Abstract

The invention provides armadillidiam vulgare plasmin and an application thereof, and relates to the field of pharmacy. The armadillidiam vulgare plasmin has thrombolytic activity and anticoagulating activity. The armadillidiam vulgare plasmin is prepared by the following steps: (1) carrying out wet ultrafine grinding on armadillidiam vulgare to obtain an extract liquid; (2) extracting a protein component with the molecular weight of 4kDa-100kDa from the extract liquid; (3) purifying the protein component with the molecular weight of 4kDa-100kDa by an ammonium sulfate precipitation method to obtain crude protein; and (4) carrying out gel filtration chromatography and anion exchange chromatography on the crude protein, and taking an eluant with thrombolytic activity to obtain the armadillidiam vulgare plasmin. According to the armadillidiam vulgare plasmin, the armadillidiam vulgare plasmin PSLTro01 is found out for the first time; and meanwhile, the armadillidiam vulgare plasmin has relatively high thrombolytic activity and anticoagulating activity, is relatively mild in thrombolytic effect, and can be used for preparing medicines for treating thrombotic diseases.

Description

technical field [0001] The invention relates to the field of pharmacy, in particular to a murine fibrinolytic enzyme and its application. Background technique [0002] Thrombotic disease is a kind of disease with high incidence and serious harm to human health, mainly including myocardial infarction, stroke, cerebral embolism, pulmonary thrombosis and so on. Thrombotic disorders are on the rise worldwide. According to statistics, an average of 12 million people die from cardiovascular and cerebrovascular diseases in the world every year, accounting for 1 / 4 of all deaths, and 80% of these deaths are in developing countries. It is estimated that by 2020, 25 million people will die of cardiovascular and cerebrovascular diseases in the world every year. In China, this proportion is even more alarming. According to the 2010 "China Health Statistics Summary", the number of deaths caused by cardiovascular and cerebrovascular diseases has exceeded 40% of the total death toll in th...

Claims

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Application Information

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IPC IPC(8): C12N9/68A61K38/48A61P7/02
CPCA61K38/00C12N9/6435C12Y304/24072
Inventor 李博吴勉华郭立玮田周
Owner NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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