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Preparation method of recombinant human granulocyte colony-stimulating factor

A technology of colony-stimulating factor and granulocytes, which is applied in the field of biomedicine, can solve the problems of low plasmid stability of bacterial strains, leaked expression of amino acid replacement impurities, etc., to improve the retention rate of plasmids, and solve the problems of low plasmid stability and amino acid replacement impurities The effect of content reduction

Active Publication Date: 2019-07-30
JIANGSU HENGRUI MEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As mentioned above, in the prior art, Escherichia coli is generally used to ferment and express recombinant human granulocyte colony-stimulating factor (rhG-CSF) on compound LB medium or self-inducing medium, which will bring about the stability of the plasmid in the fermentation process. A series of problems such as low stability, serious leakage expression and high content of amino acid replacement impurities

Method used

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  • Preparation method of recombinant human granulocyte colony-stimulating factor
  • Preparation method of recombinant human granulocyte colony-stimulating factor
  • Preparation method of recombinant human granulocyte colony-stimulating factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment one (taking 5L fermentation tank as example)

[0052] 1. Cultivation of seeds: 200mL of seed medium was placed in a 1L Erlenmeyer flask, sterilized by moist heat at 121°C for 30min, and cooled to room temperature. Take one strain of pET9a-GCSF / BL21(DE3) and inoculate it into the seed medium with an inoculum size of 0.03%, culture temperature at 30°C, shaker speed at 250rpm, shake culture for 8 hours, and measure OD 600 was 1.92, and the fermented seed culture solution was obtained. The composition and content of the seed medium are: selective soybean peptone 10g / L, yeast powder 5g / L, NaCl 5g / L, prepared with purified water, natural pH. Microscopic inspection showed that the seed culture solution was not contaminated by bacteria, which was a typical form of Escherichia coli.

[0053] 2. Preparation before inoculation: first clean the fermenter, dissolve the fermentation medium in purified water, stir evenly, transfer to the fermenter, sterilize with steam at...

Embodiment 2

[0059] Embodiment two (taking 5L fermentation tank as example)

[0060] 1. Cultivation of seeds: 200mL of seed medium was placed in a 1L Erlenmeyer flask, sterilized by moist heat at 121°C for 30min, and cooled to room temperature. Take one strain of pET9a-GCSF / BL21(DE3) and inoculate it into the seed medium with an inoculum size of 0.03%, culture temperature at 30°C, shaker speed at 250rpm, shake culture for 9 hours, and measure OD 600 2.20, to obtain a fermented seed culture solution. The composition and content of the seed medium are: selective soybean peptone 10g / L, yeast powder 5g / L, NaCl 5g / L, prepared with purified water, natural pH. Microscopic inspection showed that the seed culture solution was not contaminated by bacteria, which was a typical form of Escherichia coli.

[0061]2. Preparation before inoculation: first clean the fermenter, dissolve the fermentation medium in purified water, stir evenly, transfer to the fermenter, sterilize with steam at 121°C for 30 ...

Embodiment 3

[0066] Embodiment three (taking 5L fermentation tank as example)

[0067] 1. Cultivation of seeds: 200mL of seed medium was placed in a 1L Erlenmeyer flask, sterilized by moist heat at 121°C for 30min, and cooled to room temperature. Take one strain of pET9a-GCSF / BL21(DE3) and inoculate it into the seed medium with an inoculum size of 0.03%, culture temperature at 30°C, shaker speed at 250rpm, shake culture for 10 hours, and measure OD 600 was 2.47, and the fermented seed culture solution was obtained. The composition and content of the seed medium are: selective soybean peptone 10g / L, yeast powder 5g / L, NaCl 5g / L, prepared with purified water, natural pH. Microscopic inspection showed that the seed culture solution was not contaminated by bacteria, which was a typical form of Escherichia coli.

[0068] 2. Preparation before inoculation: first clean the fermenter, dissolve the fermentation medium in purified water, stir evenly, transfer to the fermenter, sterilize with steam...

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Abstract

The invention relates to a preparation method of a recombinant human granulocyte colony-stimulating factor. Specifically, according to the preparation method, a fermentation culture medium containingan inorganic nitrogen source and at least one amino acid is used for culturing recombinant escherichia coli expression strains. By adopting the preparation method, the stability of plasmids is high, and the yield of rhG-CSF in a fermentation liquor is high. In addition, the provided culture medium which is clear in component and simple in control strategy simplifies the process, reduces the cost,increases the protein expression quantity, improves the product quality and is beneficial to realizing industrial production.

Description

technical field [0001] The application belongs to the field of biomedicine, and in particular relates to a preparation method of recombinant human granulocyte colony-stimulating factor. Background technique [0002] Human granulocyte-colony stimulating factors (hG-CSF) are synthesized by monocyte-macrophages, vascular endothelial cells and fibroblasts. Precursor cells of phagocytes (CFU-GM) promote their differentiation and proliferation into mature granulocytes; (2) act on bone marrow mature neutrophils to promote their release from bone marrow to peripheral blood; (3) activate mature granulocytes function, enhance its migration, phagocytosis and bactericidal ability, and prolong its survival time; (4) stimulate the release of bone marrow hematopoietic stem cells into the peripheral blood. Therefore, hG-CSF is used clinically for neutrophil increase after bone marrow transplantation in cancer chemotherapy and leukemia patients. [0003] Regarding the production of hG-CSF,...

Claims

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Application Information

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IPC IPC(8): C07K14/53C07K1/107C12N15/70C12P21/02C12R1/19
CPCC07K14/53C12N15/70C12P21/02
Inventor 梅保良汪军陈磊刘衍伟王宏伟
Owner JIANGSU HENGRUI MEDICINE CO LTD
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