Supplemented culture medium of proinsulin produced by fermentation and supplemented culture optimization method

A technology of feeding medium and optimizing method, applied in microorganism-based methods, biochemical equipment and methods, fermentation, etc., can solve the problem that insulin has no biological activity, there are few studies on Escherichia coli fed batch culture, and it needs to be repeated. To solve problems such as stability, the feeding flow rate is convenient, the versatility is strong, and the expression amount is improved.

Active Publication Date: 2011-11-09
LUNAN PHARMA GROUP CORPORATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage is that the expressed insulin has no biological activity and needs renaturation
[0005] At present, researchers at home and abroad are mainly focusing on how to optimize the extraction and purification process to increase the expression of proinsulin, but there are few studies on the fed-batch culture link in the fermentation process of Escherichia coli

Method used

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  • Supplemented culture medium of proinsulin produced by fermentation and supplemented culture optimization method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] 1. Fermentation scale and various parameters

[0022] The volume of the fermenter is 50L, the host strain of E. coli BL21(DE3)PlysS is inoculated into the LB culture medium, and cultivated at 37°C for 10 hours, and it is used as the seed for the fermenter. Prepare the fermentation broth medium, make up water, sterilize at 121°C for 30 minutes, and then cool down to the optimum growth temperature of -37°C. Inoculate the seeds in a 50L fermenter according to 10% inoculum amount, and cultivate them according to the conventional method according to the growth law of the genetically engineered bacteria. Initially adjust the rotation speed to 250rpm, keep the temperature at 37°C, and adjust the air flow rate at 10L h -1 , the pH value is controlled at about 7.0, and the dissolved oxygen has been kept above 40%. Final OD of fermentation 600Set as 69, the fermentation cycle is set as 19 hours. After cultivating for two hours, take a sample to detect the cell density, continu...

Embodiment 2

[0038] Determine the initial feed flow acceleration rate V 0 =200ml·h -1 , other parameters and steps are consistent with embodiment 1.

Embodiment 3

[0040] Determine the initial feed flow acceleration rate V 0 =300ml·h -1 , other parameters and steps are consistent with embodiment 1.

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Abstract

The invention relates to a supplemented culture medium of proinsulin produced by fermentation and a supplemented culture optimization method, belonging to the field of microorganism engineering. Aiming at the defects of low fermentation density and expression amount, and the like of the prior proinsulin-presenting process by colon bacilli, the invention provides a supplemented culture medium containing a DMEM-high-glucose culture medium and a batch supplementation optimization method for applying the supplemented culture medium to proinsulin fermented by colon bacilli. The culture method has easily controlled process and strong universality and improves the supplemented culture medium of a proinsulin precursor presented by colon bacilli and the supplemented culture optimization method.

Description

technical field [0001] The invention belongs to the field of microbial engineering, and relates to a feed medium for fermenting and producing proinsulin, and also relates to an optimization method for feed culture of the medium. Background technique [0002] Diabetes is a chronic endocrine disease. As a specific drug for diabetes, insulin is used in a large amount, with a clinical dosage of several milligrams per day, and the medication takes a long time. Patients with insulin-dependent diabetes generally use insulin for life. But before 1982, all insulin used by humans was derived from animals. Porcine and bovine insulin extracted from animal organs are limited by the source of the organs, and due to the difference in the primary structure, it brings immunogenicity problems and may cause a series of side effects. In 1982, the world's first recombinant drug human insulin (reeombinanthumnainsulin, ht)l came out. Since the primary structure of recombinant human insulin is exa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/04C12R1/19
Inventor 赵志全钟传青强红刚
Owner LUNAN PHARMA GROUP CORPORATION
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