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Preparation method of recombinant hirudin

A technology for recombinant hirudin and preparation process, which is applied in the field of fermentation, can solve the problems of limited hirudin yield, cannot meet clinical application, cannot well solve the problem of high-activity hirudin preparation, etc., so as to improve expression and production efficiency. Effect

Pending Publication Date: 2020-01-14
上海松皓生物科技有限公司
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  • Abstract
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Problems solved by technology

[0003] However, the natural hirudin production is limited, and each leech only contains 20 μg of hirudin, which cannot meet the requirements of clinical application. Therefore, research on the production of recombinant hirudin by bioengineering technology—especially the production of recombinant hirudin with high yield method - it is particularly important
[0004] The recombinant hirudin prepared at present has a low expression level, which cannot well solve the problem of high-activity hirudin preparation. Therefore, the present invention provides a novel preparation process of recombinant hirudin

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  • Preparation method of recombinant hirudin

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[0019] The first aspect of the present invention provides a method for preparing recombinant hirudin, comprising: placing the seed liquid in the initial medium, and adding feed medium dropwise to the initial medium when the DO value is greater than 55%; wherein, The raw materials for the preparation of initial medium include sugar solution, trace metal solution and vitamin solution.

[0020] The applicant unexpectedly found that adding the feed medium when the DO value in the system was greater than 55% was beneficial to improving the expression activity of the obtained hirudin, and adding too early or too late had a negative impact, which would reduce the growth or The expression level of the product may be different under the condition of different DO values, and the absorption capacity of the bacteria to the feed medium is different, which will affect the expression effect of the final effective hirudin.

[0021] In a preferred embodiment, the preparation method of recombin...

Embodiment 1

[0078] Embodiment 1 of the present invention provides a recombinant hirudin, the preparation process of which includes: placing the seed liquid in the initial culture medium, passing through sterile air, and keeping the DO value greater than 40%; when the DO value is greater than 55%, the The feed medium was added dropwise to the initial medium by feeding; wherein, the ventilation rate of sterile air was 5 L / min, the temperature of the initial medium was 30°C, and the pH of the initial medium was controlled by dripping ammonia water to 5.0, and the concentration of ammonia water is 25wt%;

[0079] The preparation process of recombinant hirudin is carried out in a shaker flask, and the rotating speed is 650r / min;

[0080] The inoculum amount of seed liquid is 11%;

[0081] The preparation method of the seed liquid is: the preparation method of the seed liquid is: ① inoculate the engineering bacteria glycerol preservation bacteria into a 100mL triangular flask with 5mL seed med...

Embodiment 2

[0101] Embodiment 2 of the present invention provides a recombinant hirudin, the preparation process of which includes: placing the seed liquid in the initial culture medium, passing through sterile air, and keeping the DO value greater than 40%; when the DO value is greater than 55%, the The feed medium was added dropwise to the initial medium by feeding; wherein, the ventilation rate of sterile air was 7 L / min, the temperature of the initial medium was 30°C, and the pH of the initial medium was controlled by dripping ammonia water to 5.0, and the concentration of ammonia water is 30wt%;

[0102] The preparation process of recombinant hirudin is carried out in a shaker flask, and the rotating speed is 650r / min;

[0103] The inoculum amount of seed liquid is 11%;

[0104] The raw materials for the preparation of the initial medium include sugar solution, trace metal solution and vitamin solution;

[0105] The sugar solution includes amino-free yeast nitrogen source (YNB), ca...

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Abstract

The invention relates to the technical field of fermentation, particularly provides a preparation method of recombinant hirudin. On one hand, the invention provides the preparation method of the recombinant hirudin. The preparation method comprises the steps as follows: placing a seed solution in an initial culture medium, when the DO value is larger than 55%, dropwise adding a supplement culturemedium to the initial culture medium, wherein the preparation process of the initial culture medium comprises the steps as follows: sterilizing a sugar solution and adding to sugar solution to a fermentation tank; performing filtration sterilization on a trace metal solution, and adding the trace metal solution to the fermentation tank; and performing filtration sterilization on a vitamin solutionand adding the vitamin solution to the fermentation tank to obtain the initial culture medium.

Description

technical field [0001] The invention relates to the technical field of fermentation, and more specifically, the invention provides a preparation method of recombinant hirudin. Background technique [0002] The inhibitory effect of hirudin on thrombin is relatively independent and does not require the participation of other coagulation factors. It is selective and specific to thrombin and can directly inhibit thrombin to block its coagulation function. Hirudin is a natural inhibitor of thrombin with a single target and is relatively safe and effective among the existing known anticoagulant drugs. Hirudin can prevent the activation of coagulation factors catalyzed by thrombin and severe congestion caused by platelet reaction. Hirudin is a promising drug with significant therapeutic effects in many forms of thrombotic disease such as venous thrombosis, disseminated vascular coagulation, prophylaxis of arterial thrombosis after surgical procedures, prophylaxis after thrombolysi...

Claims

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Application Information

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IPC IPC(8): C07K14/815C12P21/02C12R1/865
CPCC07K14/815C12P21/02
Inventor 杨爱岗吴扬
Owner 上海松皓生物科技有限公司
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