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Plasminogen and cultivation method therefor

A technology for plasmin and bacterial species, applied in the field of plasmin, can solve the problems of high technical requirements, great difficulty, side effects such as bleeding, etc., and achieve the effects of low cost of raw materials and good thrombolytic performance.

Active Publication Date: 2008-03-05
QIQIHAR UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are three main treatment methods for thrombotic diseases: one is surgical operation. From the perspective of operation, this method requires high technical conditions, is also very difficult to operate, and the cost of patients is also very expensive.
The second is antithrombotic therapy, that is, long-term administration of anticoagulant drugs such as aspirin to patients. Although the disease can be controlled to a large extent, it cannot remove the already formed thrombus.
From the listing of thrombolytic drugs in the 1960s to the present, the development of thrombolytic agents can be roughly divided into three generations: the first generation of thrombolytic agents: including streptokinase (streptokinase, SK), urokinase (urokinase, UK), etc., the existing shortcomings are: This type of preparation has its own defects that are difficult to overcome, such as causing side effects such as bleeding, etc.
The second generation of thrombolytic agents: including t-PA, pro-UK, and APSAC. Although these preparations have been improved compared with the previous generation, this defect still exists
In clinical application research, it is necessary to have a large number of candidate varieties and related products for comparison and screening, but the progress we have made so far is far from meeting the needs of clinical research

Method used

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  • Plasminogen and cultivation method therefor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 1. Strain: Neurospora sitophlia No. 17 Neurospora good food

[0026] 2. Medium and culture conditions:

[0027] (1) Slant medium (PDA slant): 2% glucose, 2% agar, prepared with 20% potato juice, natural pH, cultured at 28°C for 3 days.

[0028] (2) Nystatin resistance primary screening plate medium: 10U / ml nystatin in the slant medium, 0.02% (w / v) into LiCl·H 2 O, 0.08 (w / v) sodium deoxycholate, incubated at 25-28°C for 36 hours;

[0029] (3) Solid fermentation medium for double screening: bean dregs: bran=4:1 (dry weight ratio), CaCl 2 0.75%, FeSO 4 ·7H 2 O 0.045%, natural pH, inoculum size 5×10 5 pc / bottle, ferment for 3 days at 28-30°C.

Embodiment 2

[0031] 1. Incline cultivation is the same as in Example 1

[0032] 2. Solid fermentation culture: The composition of the shake flask fermentation medium is: bean dregs: bran (4:1)+CaCl 2 0.75%+FeSO 4 ·7H 2 O 0.045%. Each bottle of medium was inoculated with 3.4×10 5 1 spore, cultivated in a 28°C incubator for 48 hours, and then leached with normal saline for 4-6 hours.

[0033] 3. Separation and purification of plasmin: After the solid fermentation product is leached with normal saline, it undergoes 40-80% saturation ammonium sulfate graded salting-out, Octyl-SepharoseFF hydrophobic interaction chromatography, SP-SepharoseHP ion-exchange chromatography, Phenyl hydrophobic interaction chromatography and RESOURCE hydrophobic interaction chromatography collect fibrinolytic active ingredients.

Embodiment 3

[0035] 1. Incline cultivation is the same as in Example 1

[0036] 2, solid fermentation is the same as embodiment 2 shallow dish fermentation;

[0037]3. Separation and purification of plasmin: Neurospora solid fermentation broth was extracted with physiological saline, 40-80% ammonium sulfate graded salting out, and Octyl-Sepharose FF hydrophobic interaction chromatography and Sephadex G-25 gel filtration were used. Chromatography, SP-SepharoseHP strong cation exchange chromatography, Superdex75 gel filtration chromatography, collected fibrinolytic active components.

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Abstract

The present invention relates to one kind of and its culturing and preparing process. The plasmin is prepared with N. sitophila strain No. 17 in the preservation number of CGMCC No. 1836, and through fermentation, separation and purification. It consists of two subunits of molecular weight 30000 and 15500 separately and isoelectric point of 7.9+ / -0.2. The plasmin has high thrombolysis performance, no bleeding activity, no blood coagulating activity and no obvious acute toxicity, and thus possesses clinical test, development and application foreground. The plasmin of the present invention is one kind of extracellular enzyme, and is favorable to post separation and purification. Its preparation adopts wheat bran and soybean residue as main culture medium material, and thus has low material cost.

Description

technical field [0001] The invention relates to a plasmin, and also relates to a method for preparing the plasmin. Background technique [0002] Thrombotic diseases seriously threaten human life and health, and are one of the main causes of human death today, and the incidence of this disease is still increasing year by year. Currently, there are three main treatment methods for thrombotic diseases: one is surgical operation. From the perspective of operation, this method requires high technical conditions, is also very difficult to operate, and the cost to patients is also very expensive. The second is antithrombotic therapy, that is, long-term administration of anticoagulant drugs such as aspirin to patients. Although the disease can be controlled to a large extent, it cannot remove the already formed thrombus. The third is thrombolytic therapy, that is, injecting thrombolytic agents to patients to promote blood vessel recanalization. Compared with the first two methods, ...

Claims

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Application Information

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IPC IPC(8): C12N9/68C12N1/14
Inventor 刘晓兰郑喜群邓永平吴耘红
Owner QIQIHAR UNIVERSITY
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