Ocean Sipunculus nudus plasmin and its preparation and application

A kind of technology of square star worm and ocean star worm, which can be applied in the fields of biochemical equipment and methods, enzymes, pharmaceutical formulations, etc., and can solve problems such as unsatisfactory

Inactive Publication Date: 2013-11-06
许瑞安
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Second-generation thrombolytics have improved, but are not

Method used

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  • Ocean Sipunculus nudus plasmin and its preparation and application
  • Ocean Sipunculus nudus plasmin and its preparation and application
  • Ocean Sipunculus nudus plasmin and its preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] 《Example 1》Preparation of sea square star worm (Sipunculus nudus) plasmin star fiber N-150 protein

[0044] Put the fresh and live marine gridworm in the seafood pool and feed it with clean seawater for 24 hours to purify the impurities in its body, wash it with phosphate buffer (0.01mol / L, pH7.4), and absorb the liquid on the surface of the insect body with filter paper Finally, dissect the parasites in a cold storage at 4°C and collect the digestive glands to measure the activity, and mix with 2 times the volume of phosphate buffer (0.01mol / L, pH7.4), after homogenization by a homogenizer, set the temperature at 10000rpm at 4°C After centrifuging for 5 minutes, the supernatant was filtered through filter paper to remove fat, and the filtrate was freeze-dried by a freeze dryer. The freeze-dried product was the crude protein powder, which was the crude extract of fibrinolytic enzyme from the sea worm.

[0045] Dissolve the lyophilized powder of the crude protein extract...

Embodiment 2

[0046] "Example 2" detection of fibrinolytic activity of star fiber N-150

[0047] According to the Astrup method and the Deogny method, namely the fibrin plate method. The specific operation is as follows: Dissolve 100mg bovine fibrinogen and 100u bovine thrombin in 10ml phosphoric acid 0.01mol / L buffer solution respectively, heat and boil 80ml 0.01mol / L phosphoric acid buffer solution containing 1g agarose to dissolve, cool to At 65°C, the bovine fibrinogen solution and the bovine thrombin solution were added to the agarose solution, stirred evenly and poured into a petri dish with a diameter of 90mm, and each petri dish contained 5ml of the mixed solution. Stand at room temperature for 30 minutes to allow it to condense. Use a puncher to punch a hole with a diameter of 3mm, spot the sample to be tested on the plate, incubate at 37°C for 4h, and measure the diameter of the dissolution circle. According to this method, the observed clear and transparent area is the area whe...

Embodiment 3

[0048] "Example 3" detection of star fiber N-150 fibrinolytic protein concentration

[0049] The determination of the fibrinolytic activity of the star fiber N-150 of the present invention is calculated based on the fibrinolytic activity of a unit protein. Star fiber N-150 fibrinolytic protein concentration is determined using Micro BCA protein assay kit. Bovine serum albumin (BSA) was used as a standard for demarcating protein concentration. Under alkaline conditions, proteins will Cu 2+ reduced to Cu + , Cu + It forms a purple complex with BCA reagent, and the absorbance intensity is proportional to the protein concentration. Measure its absorbance at 562nm and compare it with the standard curve to determine the concentration of star fiber N-150 fibrinolytic protein.

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Abstract

The invention relates to an ocean invertebrate plasmin which is a protein obtained by steps of homogenizing an ocean Sipunculus nudus digestive gland fluid, filtering to remove grease, freeze-drying, salting-out, desalinizing and carrying out DEAE chromatography and gel filtration chromatography. Amino acid sequence of the N-terminal part is Seq.2, and molecular weight is 15 KDa. It proves through experiment research that the plasmin has good effects of resisting thrombus and preventing and curing stroke.

Description

Technical field: [0001] The invention relates to marine invertebrate thrombolytic protein and its application in the treatment of cardiovascular and cerebrovascular diseases. Background technique: [0002] Thrombosis is a very important protective mechanism in the normal physiological response of an individual. When the body suffers trauma, it can effectively stop bleeding and achieve a protective effect. There are 3 main factors affecting hemostasis and thrombus formation: intravascular blood flow velocity, platelets and coagulation factors. Compared with physiological hemostasis, pathological thrombosis occurs in the vascular lumen, and if it involves the artery, it will lead to organ and tissue ischemia and / or necrosis; if it occurs in the vein, it will cause blood return obstruction. The thrombus formed in one part can also travel to blood vessels in other parts along the direction of blood flow. This process is called embolism. The inner wall of blood vessels in norma...

Claims

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Application Information

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IPC IPC(8): C12N9/68A61K38/48A61P7/02A61P9/10
Inventor 许瑞安赵永刚崔秀灵
Owner 许瑞安
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