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A kind of fibrinolytic active protein from scorpion tail and its preparation method and application

An active protein and fibrinolytic technology, applied in the preparation of anti-tumor drugs, in the field of extraction of fibrinolytic active protein from scorpion tail, can solve the problems that there are no related reports on scorpion venom fibrinolytic active protein, and achieve easy purification and batch preparation, high specific activity, and strong fibrinolytic activity

Inactive Publication Date: 2012-02-01
GUANGDONG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] So far, there has been no relevant report on the fibrinolytic activity protein of scorpion venom

Method used

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  • A kind of fibrinolytic active protein from scorpion tail and its preparation method and application
  • A kind of fibrinolytic active protein from scorpion tail and its preparation method and application
  • A kind of fibrinolytic active protein from scorpion tail and its preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Preparation of fibrinolytic active protein from scorpion venom.

[0031] Material: East Asian scorpion (origin: China)

[0032] Preparation: 1. Preparation of crude extract

[0033] Take the scorpion, cut the scorpion tail, weigh it, cut it into pieces, add pre-cooled 0.02mol / L sodium phosphate buffer pH7.2 at a ratio of 1:5 (weight: volume), grind until it is minced, and extract overnight at 4°C . Centrifuge the extract at 8500 rpm, 4°C for 15 minutes, collect the supernatant, add solid ammonium sulfate to the supernatant to reach 40% saturation, and centrifuge at 8500 rpm, 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.

[0034] 2. Ion exchange chromatography

[0035] Pre-equilibrate the DEAE-32 cellulose column with 0.02mol / L Tris-HCl buffer solution pH8.0, gradient elution with 0.02mol / ...

Embodiment 2

[0039] Determination of biological activity of plasmin in the present invention.

[0040] Reagent:

[0041] Bovine fibrinogen solution: concentration 10mg / mL. Bovine fibrinogen is a product of Sigma.

[0042] Bovine thrombin: concentration 100U / mL. Bovine thrombin is a product of Sigma.

[0043] operate:

[0044] Prepare 0.8% agarose, boil and cool to 45-55°C, add 0.8ml of 7mg / ml bovine fibrinogen, 0.5ml of 6U / ml thrombin, shake quickly and pour into a 9cm diameter petri dish, cover Put on the glass cover, cool and solidify, move to 4°C refrigerator for half an hour, use a 0.2ml pipette tip to punch holes on the prepared fibrin gel plate, add 20 μL of sample to each hole, and incubate in a 37°C incubator for 24 hours , stained with Coomassie Brilliant Blue R-250 for 30 minutes, decolorized for 2 hours, measured the size of the two vertical diameters of the dissolution circle to calculate the area of ​​the dissolution circle, and compared with the standard curve of urokina...

Embodiment 3

[0046] The application of the protein containing the fibrinolytic activity of the present invention in the preparation of antitumor drugs.

[0047] The inhibitory effect of this fibrinolytic active protein on mouse ascites tumor cell S180 was determined respectively, experiments related to intraperitoneal injection and axillary injection of solid tumor were designed, and the direct effect of this fibrinolytic active protein on S180 cells was detected by MTT assay.

[0048] 1) Establishment and results of the experimental model of intraperitoneal injection of S180 in mice

[0049] Inoculate mice (20±2g) with a cell density of 8×10 7 , intraperitoneal injection, each set 8 repeated groups, the positive control cyclophosphamide group concentration is 20mg / ml, the negative control group is equal volume of normal saline, and the drug concentration group is the above-mentioned scorpion tail fibrinolytic activity protein concentration group 25mg / ml , 100mg / ml, intraperitoneal injectio...

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Abstract

The invention discloses a fibrinolytic active protein from scorpion tail and its preparation method and application. The preparation method of the fibrinolytic active protein with a molecular weight of 37.69kD from scorpion tail is as follows: washing the scorpion tail, grinding crushed, added to sodium phosphate buffer, leached overnight at 4°C; centrifuged to remove the precipitate, salted out in sections, and 40%-70% of the fraction was taken; finally purified by diethylaminoethyl-32 cellulose ion-exchange chromatography column, and used Elution with Tris-HCl buffer solution, collect active protein peaks, concentrate, and freeze-dry; the application of the fibrinolytic active protein of the present invention in the preparation of antitumor drugs; the fibrinolytic active protein involved in the invention is a single group in scorpion tail It is easy to purify and prepare in batches, has strong fibrinolytic activity, high specific activity, and no toxicity; it has small molecular weight and low antigenic reaction, and is an ideal antitumor drug.

Description

technical field [0001] The invention relates to the field of biology, in particular to the extraction of fibrinolytic active protein in scorpion tail. The invention extracts a fibrinolytic active protein from scorpion tail, relates to a preparation method of the fibrinolytic active protein, and an application of the fibrinolytic active protein in preparing antitumor drugs. Background technique [0002] Scorpions are one of the oldest species in evolution, with a history of 400 million years ago. During the long evolutionary process, their shape has hardly changed. There are about 1,500 kinds of scorpions around the world, which are divided into 6 families. The reason why scorpions have aroused great interest of scientists is not only because scorpions have experienced a long evolutionary process, but more importantly, because the scorpion venom secreted by the scorpion tail gland has extremely strong great medical value. Scorpion venom contains a variety of biologically ac...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C07K1/36C07K1/34C07K1/30C07K1/18A61K38/17A61P35/00
Inventor 谭竹钧黄锦兵韩雅莉陈雅雄丁鸿
Owner GUANGDONG UNIV OF TECH
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