Marine phasolosma esculenta fibrinolytic enzyme, preparation method and applications thereof

A kind of Delicious Cosmia and plasmin technology, applied in the application of stroke and anti-tumor, treatment of thrombosis, the field of preparation of marine Delicious Cosmia plasmin, can solve problems such as unsatisfactory

Inactive Publication Date: 2013-10-02
许瑞安
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Second-generation thrombolytics have improved, but are not ideal
The results of this study have not been reported so far

Method used

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  • Marine phasolosma esculenta fibrinolytic enzyme, preparation method and applications thereof
  • Marine phasolosma esculenta fibrinolytic enzyme, preparation method and applications thereof
  • Marine phasolosma esculenta fibrinolytic enzyme, preparation method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] 《Example 1》Preparation of Plasmina plasmolytic enzyme from the sea

[0040] Experimental Materials : Phascolosoma esculenta (Phascolosoma esculenta), purchased from Anhai City, Jinjiang, Fujian Province.

[0041] preliminary separation : Put the fresh and delicious Dermocystus in the seawater pool for a few days, and excrete the visceral impurities. According to the results of the active site detection, take the yellow intestinal tissue, add a certain volume of 0.02mol / L Tris-HCl buffer solution (pH7.4), cut it with scissors, put it in the refrigerator, and store it between -20°C and 4°C Repeated freezing and thawing 3 times. Put the tissue into the homogenizer for a few seconds, take it out and centrifuge at 10,000 rpm for 10 min at 4°C to remove the precipitate. Measure the volume of the supernatant and mix it with saturated ammonium sulfate of pH 7.4 to make the concentration of ammonium sulfate in the solution reach 30%, place at 4°C for 12h. Then refrigerate...

Embodiment 2

[0051] "Example 2" measures the method for the activity of Plasmodium pallidum fibrinolytic enzyme:

[0052] Slightly improved with reference to the Astrup fibrin plate method, its specific operation process: Weigh 1g of agarose and place it in a 100mL Erlenmeyer flask, add 45mL of 0.02mol / L Tris-HCl buffer solution (pH7.4) to prepare 2% For the agar solution, boil the water bath until the agarose is completely melted, then put it in a constant temperature water bath at 55°C. Take 1mL of thrombin (100U / mL) and dilute it to 2.5mL, weigh 50mg of fibrin and dissolve it in 2.5mL of 0.02mol / L Tris-HCl buffer (pH7.4). Add the thrombin and fibrin solution to the agarose in a water bath at 55°C, stir evenly, pour it into a sterilized 9cm petri dish, and cool it at room temperature to form a gel, which is a standard plate. A hole with a diameter of 0.5 cm was punched on the plate with a puncher.

[0053] Plasmin activity assay :

[0054] Make a standard plate, punch 7 holes with a...

Embodiment 3

[0055] "Example 3" Determination of the N-terminal Amino Acid Sequence of the PFE N-terminal Amino Acid Sequence

[0056] After SDS-PAGE electrophoresis, use 10×CAPS transfer buffer (100mmol / L) for protein transfer: Take 22.13g of CAPS, add 900mL of deionized water, adjust the pH to 11.0 with 2mol / L NaOH, and then constant volume to 1L and stored at 4°C. When transferring, take 200mL of 10×CAPS transfer buffer, 200mL of methanol, and 1600mL of deionized water. Transfer process: put the gel and PVDF membrane in a transfer tank containing pre-cooled transfer buffer, and transfer for 1.5h under the condition of a voltage of 200V. Take out the PVDF membrane and rinse it slightly with deionized water, soak it in methanol for a few seconds, then stain it with 0.1% Coomassie Brilliant Blue R-250, decolorize it with 50% methanol, and wash it thoroughly with deionized water. The purified enzyme components were subjected to SDS-PAGE electrophoresis, showing a single protein band aroun...

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Abstract

The invention relates to fibrinolytic enzyme separated and prepared from a marine invertebrate, wherein 7 amino acid sequences on the N-terminal are NH2-IVGGRDV-OH, the molecule weight is about 35 kDa, and experimental study results show that the fibrinolytic enzyme and drug compositions thereof provide significant effects for anti-thrombus and stroke prevention and treatment, and further provide good effects in the field of tumor treatment.

Description

Technical field: [0001] The invention relates to a marine invertebrate thrombolytic protein and its preparation and application, in particular, to the preparation of a marine D. cactus fibrinolytic enzyme and its application in treating thrombus, stroke and antitumor. Background technique: [0002] Thromboembolic diseases mainly include two types: arterial thromboembolic diseases and venous thromboembolic diseases, among which acute arterial thrombosis is the main cause of myocardial infarction, and more than 80% of strokes are also caused by it. Venous thromboembolism is the third leading cause of death from cardiovascular disease. [0003] The main trigger of arterial thrombosis is the rupture of atherosclerotic plaque, which is formed by the accumulation of lipid deposits and foam cells on the artery wall. Ruptured plaques are rich in platelets derived from bone marrow megakaryocytes, which function to rapidly accumulate in the blood as initial hemostatic plugs appear at...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/68A61K38/48A61P7/02A61P9/10A61P35/00
Inventor 许瑞安熊亚楠崔秀灵
Owner 许瑞安
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