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Yellow mealworm plasmin and preparation method and applications thereof

A technology for plasmin and Tenebrio molitor, applied in the biological field, can solve the problems of high price, no Tenebrio plasmin, short half-life, etc., and achieves the effects of high specific activity, easy purification and batch preparation, and small molecular weight

Inactive Publication Date: 2011-11-09
GUANGDONG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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
So far, there is no related report about Tenebrio plasmin

Method used

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  • Yellow mealworm plasmin and preparation method and applications thereof
  • Yellow mealworm plasmin and preparation method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Preparation of Tenebrio molitor fibrinolytic enzyme.

[0028] Material: Tenebrio molitor (origin: China)

[0029] Preparation: 1. Preparation of crude extract

[0030] Weigh 10g of Tenebrio molitor, cut it into pieces, add 50mL of pre-cooled 0.02mol / L sodium phosphate buffer solution (pH7.4~8.5) at a ratio of 1:5 (weight: volume), grind until it becomes a paste, and extract at 4°C overnight. Centrifuge the extract at 8,500 rpm, 4°C for 15 minutes, and collect the supernatant; add solid ammonium sulfate to the supernatant to reach 30% saturation, and centrifuge at 8,500 rpm, at 4°C , collect the supernatant; then add solid ammonium sulfate to reach 70% saturation, 8500 rpm, centrifuge in a centrifugal pump at 4°C, collect the precipitate and carry out dialysis concentration to obtain a crude extract.

[0031] 2. Ion exchange chromatography

[0032] Balance the DEAE-cellulose 32 ion-exchange chromatography column with 0.02mol / L pH8.0 Tris-HCl buffer in adva...

Embodiment 2

[0037] Example 2: Determination of the biological activity of plasmin in the present invention.

[0038] The assay method of plasmin 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:

[0039] Reagent: bovine fibrinogen solution: concentration 10mg / mL. Bovine fibrinogen is a product of Sigma.

[0040] Bovine thrombin: concentration 100U / mL. Bovine thrombin is a product of Sigma.

[0041] operate:

[0042] 1. Preparation of fibrinolytic plates

[0043] First prepare 10ml of 0.8% agarose, boil, cool to 45-55°C, add 0.25ml of 10mg / ml bovine fibrinogen and 0.025ml of 100U / ml thrombin, shake quickly, and pour into a culture medium with a diameter of 9cm In a dish, cover with a glass lid, cool and solidify (about 5 minutes), then transfer to a 4°C refrigerator for half an hour.

[0044...

Embodiment 3

[0046] Example 3: Preparation of a pharmaceutical composition containing plasmin of the present invention.

[0047] Get 50 micrograms of fibrinolytic enzyme of the present invention, dissolve in 5 milliliters of distilled water. Seal and store at room temperature.

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Abstract

The invention discloses a yellow mealworm plasmin and a preparation method and applications thereof, the plasmin is extracted from yellow mealworm and the molecular weight thereof is 56.1 kilodalton, and the invention also relates to the preparation method of the yellow mealworm plasmin and the applications thereof in preparing medicaments for treating thrombus; the preparation method comprises the steps of cleaning and weighting the yellow mealworm, adding sodium phosphate buffer solution, grinding the yellow mealworm to be mash, extracting at 4 DEG C and staying overnight; centrifuging to remove the sediment, adding solid ammonium sulfate into supernate and conducting fractional salting out, taking 30%-70% of saturation salting out components for dialysis and concentration, thus obtaining crude extraction; and purifying the crude extraction by a diethylaminoethyl-cellulose 32 ion exchange chromatographic column, using Tris-HCl buffer solution for gradient elution, and collecting highly abundant protein peaks. The plasmin is easy for purification and batch preparation, has strong plasmin activity, high specific activity, no toxicity, small molecular weight and low antigen reaction, and is comparatively ideal plasmin preparation material and an ideal thrombolytic drug.

Description

technical field [0001] The present invention relates to the biological field, and the present invention extracts a kind of fibrinolytic enzyme from Tenebrio molitor, and relates to the preparation method of this fibrinolytic enzyme, the composition containing this kind of fibrinolytic enzyme, and this kind of fibrinolytic enzyme in the preparation of therapeutic blood Use in medicine for embolic diseases. Background technique [0002] At present, the incidence of blood embolism diseases caused by various reasons, such as cerebral thrombosis, myocardial infarction, coronary heart disease, limb vascular embolism, etc., is on the rise; only our country spends hundreds of billions of dollars on prevention and rehabilitation every year Such diseases seriously endanger the quality of life of patients and increase the burden on patients' families and society. Therefore, the research and development of clinical drugs for such diseases is also progressing rapidly; clinically more co...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/68A61K38/48A61P7/02
Inventor 谭竹钧陈雅雄韩雅莉黄锦兵
Owner GUANGDONG UNIV OF TECH
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