Recombinant human insulin genetically engineered bacterium high expression strain selection culture medium and preparation method

A technology of recombinant human insulin and genetically engineered bacteria, which is applied in the direction of insulin, microbial-based methods, biochemical equipment and methods, etc., can solve the problems of single nutrient composition in the culture medium, increased pollution probability, complicated screening process, etc., to reduce pollution , screening stability, and improved screening method performance

Active Publication Date: 2016-06-22
TONGHUA DONGBAO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, recombinant human insulin genetically engineered bacteria have defects such as single nutrient composition in the medium, low expression level, a

Method used

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  • Recombinant human insulin genetically engineered bacterium high expression strain selection culture medium and preparation method
  • Recombinant human insulin genetically engineered bacterium high expression strain selection culture medium and preparation method
  • Recombinant human insulin genetically engineered bacterium high expression strain selection culture medium and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] according to figure 1 Precisely prepare 1 liter of recombinant human insulin genetically engineered bacteria high-expression strain screening medium, each component and its quality are: peptone 1.5g, yeast powder 3g, potassium dihydrogen phosphate 1.5g, disodium hydrogen phosphate 4.5g , sodium chloride 0.3g, ammonium chloride 0.1g, adjust the pH to 6.80-7.20, and the balance is purified water.

[0034] Weigh the raw materials of each component according to the stated ratio, dissolve them in purified water to 90% of the final volume, adjust the pH to 6.80-7.20, after constant volume, sterilize at 121.0°C for 30 minutes, add sterile kanamycin to a final concentration of 40mg / L.

[0035] Prepare the screening medium for the high-expression strain of recombinant human insulin genetically engineered bacteria, using existing recombinant human insulin genetically engineered bacteria (host bacteria is E.colik12W3110, expression plasmid is PL-82, containing kanamycin (Kana) a...

Embodiment 2

[0037] according to figure 1 According to the process, 1 liter of recombinant human insulin genetically engineered bacteria high-expression strain screening medium was precisely prepared. The components and their quality were: 3g of peptone, 6g of yeast powder, 3.5g of potassium dihydrogen phosphate, 6.5g of disodium hydrogen phosphate, Sodium chloride 0.6g, ammonium chloride 0.3g, adjust the pH to 6.80-7.20, and the balance is purified water.

[0038] Weigh the raw materials of each component according to the stated ratio, dissolve them in purified water to 90% of the final volume, adjust the pH to 6.80-7.20, after constant volume, sterilize at 121.0°C for 30 minutes, add sterile kanamycin to a final concentration of 30mg / L.

[0039] Prepare the screening medium for the high-expression strain of recombinant human insulin genetically engineered bacteria, using the existing recombinant human insulin genetically engineered bacteria (host bacteria is E.colik12W3110, expression ...

Embodiment 3

[0041] according to figure 1 According to the process, 1 liter of recombinant human insulin genetically engineered bacteria high-expression strain screening medium was precisely prepared. The components and their mass fractions were: peptone 2g, yeast powder 5g, potassium dihydrogen phosphate 2.5g, disodium hydrogen phosphate 5g, chlorine 0.5 g of sodium chloride and 0.2 g of ammonium chloride were used to adjust the pH to 7, and the balance was purified water.

[0042] Weigh the raw materials of each component according to the stated ratio, dissolve them in purified water to 90% of the final volume, adjust the pH to 6.80-7.20, after constant volume, sterilize at 121.0°C for 30 minutes, add sterile kanamycin to a final concentration of 40mg / L.

[0043] Prepare the screening medium for the high-expression strain of recombinant human insulin genetically engineered bacteria, using the existing recombinant human insulin genetically engineered bacteria (host bacteria is E.colik12...

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Abstract

The invention relates to a microorganism selection culture medium. The culture medium is a liquid culture medium. The culture medium comprises the components: peptone, yeast powder, monopotassium phosphate, disodium hydrogen phosphate, sodium chloride, ammonium chloride, antibiotics and the balance of water. The culture medium is comprehensive in nutrition, simple in preparation and low in cost and has the advantage that the engineered bacterium strain with stability and high expression quantity can be selected. By adopting the culture medium, the selection flow of the recombinant human insulin genetically engineered bacterium high-expression strain is optimized, and an inductive agent does not need to be added in the middle of the strain culture period, so that the selection flow is simplified, and the pollution risk is reduced. Therefore, the culture medium and the preparation method are suitable for the industrialized production of the recombinant human insulin.

Description

technical field [0001] The invention belongs to the field of microbial strain screening, and in particular relates to a screening medium for recombinant human insulin genetically engineered bacteria high-expression strains and a preparation method thereof. Background technique [0002] In recent years, with the improvement of people's living standards, the incidence of diabetes has been increasing year by year. Diabetes is the third leading cause of death after cardiovascular and tumors. According to the data released by the International Diabetes Federation (IDF), in 2014 The number of people with diabetes worldwide (aged 20 to 79) reached 387 million in 2019, an increase of 5 million from last year, and is expected to reach 592 million in 2035. A large number of clinical practices have confirmed that the treatment of diabetes with recombinant human insulin injection can achieve ideal results. [0003] Escherichia coli E.coli is a commonly used prokaryotic expression syste...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12R1/19
CPCC07K14/62C12N1/20
Inventor 冷春生李一奎王明焱周芳常晓慧
Owner TONGHUA DONGBAO PHARMA
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