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Fibrinolytic activated protein from cantharis, preparation method and application thereof

A technology of active protein and fibrinolysis, applied in the application of medicine, in the field of extraction of fibrinolytic active protein in Cantharidin, can solve the problems of low antigen response, short half-life, high price, etc. live high effect

Inactive Publication Date: 2011-01-19
GUANGDONG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Second-generation preparations such as recombinant tissue plasminogen activator (rt-PA), p-methoxybenzoyl plasminogen streptokinase complex (APSAC) and recombinant streptokinase (r-SK), etc. The main feature is selective thrombolysis, which has less effect on systemic fibrinolytic activity, but its half-life is short and expensive
[0013] But still do not see the related report about mylabris fibrinolytic activity protein so far, the fibrinolytic protein that the present invention relates to is a kind of single component protein in the mylabris body, and molecular weight is at 95.5kD, is a kind of new fibrinolytic protein, is easy to purify And batch preparation, and strong fibrinolytic activity, high specific activity, non-toxic; small molecular weight, low antigenic reaction, is an ideal raw material for fibrinolytic enzyme preparations, is an ideal thrombolytic drug

Method used

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  • Fibrinolytic activated protein from cantharis, preparation method and application thereof
  • Fibrinolytic activated protein from cantharis, preparation method and application thereof
  • Fibrinolytic activated protein from cantharis, preparation method and application thereof

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Embodiment 1

[0032] The present invention can be described in detail below by way of examples, but these examples do not limit the scope of the present invention in any way. Embodiment 1: Preparation of mylabris fibrinolytic activity protein

[0033] Material: Mylabris (origin: China)

[0034] preparation:

[0035] 1. Preparation of crude extract

[0036] Weigh 10g of cantharidin, cut it into pieces, add 100mL of pre-cooled 0.02mol / L sodium phosphate buffer solution (pH7.4) at a ratio of 1:10 (dry weight: volume), soak for 2h, grind until it becomes minced, and store at 4°C Leach overnight. Centrifuge the extract in a high-speed centrifuge at 8,000 rpm at 4°C for 15 minutes, collect the supernatant, add solid ammonium sulfate to the supernatant to reach 20% saturation, and centrifuge at 8,000 rpm at 4°C Centrifuge in the machine for 30min, collect the supernatant; then add solid ammonium sulfate to reach 80% saturation, centrifuge in a high-speed centrifuge at 4°C for 30min at 8000 rpm...

Embodiment 2

[0041] Embodiment 2: Determination of biological activity of mylabris fibrinolytic activity protein of the present invention

[0042] The assay method of fibrinolytic activity is well known to those skilled in the art. Because the fibrinolytic plate method is highly sensitive and can be both qualitative and quantitative, the fibrinolytic plate method was used in this study. The specific methods are as follows:

[0043]Reagent: bovine fibrinogen solution, concentration 10mg / mL, bovine fibrinogen is a product of Sigma; bovine thrombin, concentration 100U / mL, bovine thrombin is a product of Sigma; urokinase, urokinase is a product of Sigma .

[0044] operate:

[0045] To prepare a fibrin plate, dissolve 0.1g of agarose in 12mL of phosphate buffer (PBS, pH=7.6), boil, cool and keep warm in a water bath at 50°C, add 0.5mL of fibrinogen solution (10mg / ml) and 20μL of thrombin (100u / ml) solution, after mixing, quickly pour it into a 50°C preheated petri dish (diameter 9cm), let it...

Embodiment 3

[0046] Embodiment 3: In vitro thrombolysis experiment and hemolysis experiment containing fibrinolytic active protein of the present invention

[0047] The in vitro thrombolytic experiment uses mouse thrombus as a model to determine the in vitro thrombolytic effect of the fibrinolytic active protein of the present invention; and the hemolytic experiment is an important indicator for investigating the safety of injections, and the mouse blood is used as a model to determine the fibrinolytic activity of the present invention. Whether the administration of protein as an injection for the treatment of thrombus will cause a hemolytic reaction; the above experimental method is well known to those skilled in the art, and is easy to operate and convincing. The specific method is as follows:

[0048] Reagents: mouse blood (blood was taken from the orbit of mice used for experiments); urokinase, urokinase is a product of Sigma Company.

[0049] operate:

[0050] Thrombolytic experiment...

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Abstract

The invention discloses a fibrinolytic activated protein from cantharis, a preparation method and an application thereof. In the invention, the single-component protein with fibrinolytic activity is extracted from the cantharis, and the molecular weight of the protein is 95.5KD; and the protein has the advantages of easy purification and batch preparation, stronger fibrinolytic activity, high specific activity, no toxicity, small molecular weight and low antigen reaction, thus being an ideal plasmin preparation raw material as well as an ideal thrombolytic drug. The invention further discloses the preparation method of the fibrinolytic activated protein and the application of the same for preparing the drug for treating thrombotic disease.

Description

technical field [0001] The invention relates to the field of biology, in particular to the extraction of fibrinolytic active protein in mylabris. The invention extracts a single-component protein with fibrinolytic activity from mylabris, relates to a preparation method of the fibrinolytic active protein, and an application of the fibrinolytic active protein in preparing medicine for treating blood embolism diseases. Background technique [0002] Mylabris is an insect of the family Carthariaceae, and its application has been recorded in works such as "Shen Nong's Herbal Classic" and "Compendium of Materia Medica" in my country. "Shen Nong's Materia Medica" records that mylabris is very poisonous, and it returns to the liver, stomach, kidney, large intestine and small intestine meridian. [0003] At present, the incidence of blood embolism diseases caused by various reasons, such as cerebral thrombosis, myocardial infarction, coronary heart disease, limb vascular embolism, et...

Claims

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Application Information

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IPC IPC(8): C07K14/745C07K1/36C07K1/30C07K1/18A61K38/36A61P7/02
Inventor 谭竹钧汪威韩雅莉吴艳玲
Owner GUANGDONG UNIV OF TECH
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