Method for preparing recombinant human octoplasmin

A technology of plasmin and plasminogen, which is applied in the field of biomedicine, can solve the problems of staphylokinase rarity and high production cost, and achieve the effects of increasing expression, simple operation, and reducing degradation

Active Publication Date: 2022-05-13
JINGZE PHARMA (HEFEI) CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, natural staphylokinase is very rare, and the preparation cost is very high. In order to reduce the production cost, the industry mainly uses recombinant staphylokinase as the plasminogen activator to prepare plasmin

Method used

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  • Method for preparing recombinant human octoplasmin
  • Method for preparing recombinant human octoplasmin
  • Method for preparing recombinant human octoplasmin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1: The process of fermenting recombinant human Oak plasminogen

[0074] The processing steps of the present embodiment are as follows:

[0075] 1. Preparation of seed solution

[0076] The glycerin seeds of engineering bacteria were thawed, inoculated into BMGY medium shake flasks and amplified after shaking culture to obtain OD 600 Seed solution with a value between 5.0 and 15.0.

[0077] 2. Fermentation culture in fermenter (batch 32F171104)

[0078] Inoculate the seed liquid prepared above to BSM basal medium for fermentation (the volume of the inoculated seed liquid is 10% of the volume of the BSM basal medium), the culture temperature is 30.0±1.0°C, the pH of the fermented liquid is controlled at 5.0±0.5, and the dissolved oxygen is maintained. (D.O) is above 10%. Cultivate until the initial glycerol in the fermentation broth is exhausted (dissolved oxygen in the fermentation broth rises to 100% at this time), supplement 50% glycerol with 3% of the init...

Embodiment 2

[0084] Example 2: The process of fermenting recombinant human Oak plasminogen

[0085] The processing steps of the present embodiment are as follows:

[0086] 1. Preparation of seed solution

[0087] The glycerin seeds of engineering bacteria were thawed, inoculated into BMGY medium shake flasks and amplified after shaking culture to obtain OD 600 Seed solution with a value between 5.0 and 15.0.

[0088] 2. Fermentation tank culture (32F171105)

[0089] Inoculate the seed solution prepared above into BSM medium for fermentation (the volume of the inoculated seed solution is 10% of the volume of the BSM basal medium), the culture temperature is 30.0±1.0°C, the pH of the fermentation solution is controlled at 5.0±0.5, and the D.O is maintained at 10 %above. Cultivate until the initial glycerol in the fermentation broth is exhausted (dissolved oxygen in the fermentation broth rises to 100% at this time), replenish 50% glycerol with an initial fermentation broth volume of 6.25...

Embodiment 3

[0093] Example 3: Process for fermenting recombinant human Oak plasminogen

[0094] 1. Preparation of seed solution

[0095] The glycerin seeds of engineering bacteria were thawed, inoculated into BMGY medium shake flasks and amplified after shaking culture to obtain OD 600 Seed solution with a value between 5.0 and 15.0.

[0096] 2. Fermentation tank culture (32F171106)

[0097]The fermentation base medium is BSM medium, the culture temperature is 30.0±1.0°C, the pH of the fermentation broth is controlled at 5.0±0.5, and the D.O is maintained above 10%. Cultivate until the initial glycerol in the fermentation broth is exhausted (dissolved oxygen in the fermentation broth rises to 100% at this time), supplement 50% glycerol with an initial fermentation broth volume of 8.3%, and stop when the cell wet weight in the fermentation broth reaches 185g / L Add glycerin. When the added glycerin in the fermentation broth is exhausted (the dissolved oxygen in the fermentation broth ri...

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Abstract

The invention provides a method for preparing recombinant human octoplasmin, and belongs to the technical field of biological medicine. The method comprises the following steps: (1) preparing staphylokinase subjected to enzyme digestion: taking recombinant staphylokinase for enzyme digestion, and separating and purifying an enzyme digestion substance to obtain staphylokinase subjected to enzyme digestion; wherein the amino acid sequence of the staphylokinase subjected to enzyme digestion is as shown in SEQ ID NO: 3; and (2) preparing the recombinant plasmin: adding staphylokinase subjected to enzyme digestion into the recombinant plasminogen for enzyme digestion, and separating and purifying an enzyme digestion substance to obtain the recombinant plasmin. It is found for the first time that the impurity A in the recombinant plasmin can be effectively removed through the method, the purity of the recombinant plasmin is improved, and potential stability and safety risks of drugs in clinical application are eliminated. The method disclosed by the invention is low in cost, simple to operate and suitable for industrial production.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for preparing recombinant human aukiplasmin. Background technique [0002] Thrombosis in the human body often leads to severe cardiovascular diseases, which are sudden onset and extremely high in mortality. Thrombolytic therapy is a common method for treating such diseases. Human plasminogen is one of the key components of the human fibrinolytic system. The plasmin obtained by activating human plasminogen with a plasminogen activator has serine protease activity and can degrade various proteins including laminin, fibronectin and collagen. Studies have found that plasmin can separate the vitreous from the retina by hydrolyzing the adhesion molecules between the posterior vitreous membrane and the retinal basement membrane, thereby achieving the purpose of treating vitreomacular adhesions and macular traction. [0003] Human plasmin consists of 791 amino ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/68
CPCC12N9/6435C12Y304/21007Y02A50/30
Inventor 彭红卫李宁
Owner JINGZE PHARMA (HEFEI) CO LTD
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