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Activator protein for fibrinolytic system

A technology of fibrinolysis and protein activation, applied in the fields of peptide/protein components, cardiovascular system diseases, anti-animal/human immunoglobulin, etc., can solve the problem of thrombolytic active components without further progress, earthworm medicinal function Restrictions and other issues, to achieve the improvement of hemorheology indicators, good economic value and social benefits, and promote the effect of thrombolysis

Active Publication Date: 2010-10-13
成都瑞益科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no further progress has been made in the final determination of the thrombolytically active components in earthworms
The further application of the medicinal function of earthworms is limited

Method used

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  • Activator protein for fibrinolytic system
  • Activator protein for fibrinolytic system
  • Activator protein for fibrinolytic system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Separation and purification of FAP in earthworms

[0054] Materials and Methods

[0055] Earthworms: Assay of FAP activity of earthworms (Eisenia fortida) cultured in the suburbs of Wuxi: using Example 3 (FAP in vitro plasminase activity and plasminogen kinase activity), Example 4 (FAP on photochemically induced rats) The dissolution of focal cerebral thrombus), the method described in Example 5 (preparation and application of FAP antibody) is a detection means to measure the biological activity and immune activity of FAP.

[0056] FAP extraction: wash the fresh earthworm tissue, after homogenization, add the same amount of water, extract at 25°C for 12-14h, centrifuge at 8000r / min, supernatant is salted out with 20% and 50% ammonium sulfate, and take 50% The salting-out precipitate was dialyzed against water overnight, the dialysate was centrifuged at 17000r / min, and the supernatant was freeze-dried. Extract A is obtained.

[0057] Gel filtration: Sephadex...

Embodiment 2

[0062] In vitro recombinant expression of embodiment 2 FAP

[0063] 1. FAP gene acquisition

[0064] A. Earthworm mRNA Extraction

[0065] (1) Weigh 0.2g fresh earthworm (Eisenia fortida), add 1ml TRIzol reagent to prepare earthworm homogenate, and incubate at 4°C for 5min.

[0066] (2) Add 0.2ml of chloroform, close the cap tightly and shake vigorously for 15Sec, then place it on ice for 5min.

[0067] (3) 4°C, 12000×g, centrifuge for 15 minutes.

[0068] (4) Transfer the upper aqueous phase to another tube, add 0.5ml of isopropanol, and incubate on ice for 10min.

[0069] (5) Centrifuge at 12000×g for 10 minutes at 4°C.

[0070] (6) Discard the supernatant, add 1 ml of 75% ethanol to the precipitate (containing RNA) to wash, and vortex to mix.

[0071] (7) Centrifuge at 10,000×g for 5 minutes at 4°C to obtain RNA precipitates.

[0072] (8) After air drying, dissolve with appropriate amount of TE or RNase-free water for later use.

[0073] B. Primer Design

[0074] Pr...

Embodiment 3

[0129] In vitro plasmin activity and plasminogen kinase activity assay of embodiment 3 FAP

[0130] Materials and Methods

[0131] Materials: FAP (purified from earthworms); plasminogen (bovine blood, each tube is equivalent to 12 casein units); thrombin (bovine blood, each tube is equivalent to 130 BP units), urokinase (each tube is equivalent to 730 units) and Fibrinogen (coagulable protein 81mg per vial) (China, National Institute for the Control of Pharmaceutical and Biological Products)

[0132] The fibrin plate method is used to measure the kinase activity and plasmin activity of FAP. According to the fibrin plate method of Doegny et al., slightly improved:

[0133] 1) Determination of plasminogen kinase activity: 200 μl plasmin stock solution + 100 μl thrombin (bovine blood). After mixing, add it to the fibrin stock solution preheated at 37°C, pour the mixed solution into the agar solution at 56°C, mix quickly, spread two flat plates with a diameter of 9 cm, place the...

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Abstract

The invention provides an activator protein for fibrinolytic system, which is: 1) a protein formed by an amino acid sequence as shown in SEQ ID No.2, or 2) a protein which is formed by substituting, losing and / or adding one or a plurality of amino acids to the amino acid sequence as shown in SEQ ID No.2, and is derived from 1) and has the equivalent functions. Experiments show that the protein can obviously enhance the in vivo fibrinolysis effect, promote thrombolysis, and obviously improve hemorheological indicators. Besides, the protein does not affect the blood coagulation system and anticoagulation indicators, and is an ideal medicament for treating or preventing diseases or microcirculatory disturbances caused by thrombosis. The activator protein for the fibrinolytic system has wide application prospects and favorable economic value and social benefits.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a protein capable of activating a fibrinolytic system, which can be used for treating and preventing diseases related to thrombosis and microcirculation disturbance. Background technique [0002] From the 1980s to the early 1990s, a global aging society emerged, and the number one killer that endangers the lives of middle-aged and elderly people is cardiovascular and cerebrovascular diseases. According to the statistics of the World Health Organization (WHO), about 12 million people die of cardiovascular and cerebrovascular diseases in the world every year. The incidence of stroke in China is about 219 / 100,000, and the prevalence rate is 761 / 100,000. About 9 million people in the country suffer from stroke, and about 2 / 3 of the number of patients are ischemic strokes. The formation of thrombus is the cause of stroke. The main cause of ischemic stroke and other cardiovascular and...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K1/36C07K1/30C07K1/26C07K1/18C07K1/16C07K16/18C12N15/12C12N15/63G01N33/53A61K38/17A61P7/02A61P9/10
Inventor 张益民王凌
Owner 成都瑞益科技有限责任公司
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