Plasmin cultivation method

A technology of plasminase and bacteria strains, applied in the field of plasminase cultivation, can solve the problems of high technical requirements, great difficulty, and inability to meet research needs, and achieve low cost of raw materials and good thrombolytic performance Effect

Active Publication Date: 2010-05-19
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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0026] 2. Medium and culture conditions:

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

[0028] (2) Preliminary screening plate medium for Nystatin resistance: Inclined medium contains 10U / ml Nystatin, 0.02% (w / v) into LiCl·H 2 O, 0.08 (w / v) sodium deoxycholate, culture at 25~28℃ for 36h;

[0029] (3) Solid fermentation medium for re-screening: bean dregs: bran = 4:1 (dry weight ratio), CaCl 2 0.75%, FeSO 4 ·7H 2 O 0.045%, natural pH, inoculation amount is 5×10 5 Pieces / bottle, fermentation at 28~30℃ for 3d.

Embodiment 2

[0031] 1. Inclined 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%. Inoculate 3.4×10 per bottle of medium 5 The spores were cultured in a thermostat at 28°C for 48 hours, and then extracted with physiological saline for 4-6 hours. The composition and culture conditions of the shallow plate fermentation medium were the same as those in the shake flask culture.

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

Embodiment 3

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

[0036] 2. The solid fermentation is the same as that of Example 2 shallow pan fermentation;

[0037] 3. Separation and purification of fibrinolytic enzyme: Neurosporum solid fermentation broth is leached in physiological saline, 40-80% ammonium sulfate fractional salting out, Octyl-Sepharose FF hydrophobic interaction chromatography, Sephadex G-25 gel filtration Chromatography, SP-SepharoseHP strong cation exchange chromatography, Superdex75 gel filtration chromatography, collect 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 throughfermentation, 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 method for culturing plasmin. Background technique [0002] Thrombosis diseases seriously threaten human life and health, and are one of the main causes of human deaths today, and the incidence of this disease is still increasing year by year. There are three main treatment methods for thrombotic diseases at the moment: one is the surgical method. From the operation point of view, this method requires high technical conditions, the operation is also very difficult, and the costs that patients need to pay are also very expensive. The second is antithrombotic therapy, which is to give patients long-term anticoagulant drugs such as aspirin. Although the disease can be controlled to a large extent, it cannot remove the formed thrombus. The third is thrombolytic therapy, that is, injecting thrombolytic agents into patients to promote vascular recanalization. Compared with the previous two methods, this method is highly efficient, spec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/68C12N1/14C12R1/645
Inventor 刘晓兰郑喜群邓永平吴耘红
Owner QIQIHAR UNIVERSITY
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