A kind of 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 strains, leaked expression of amino acid replacement impurity content, etc., achieve strong plasmid stability, solve leaked expression phenomenon, and stable and controllable fermentation process Effect
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Embodiment 1
[0051] Embodiment 1 (take 5L fermentation tank as an example)
[0052] 1. Cultivation of seeds: 200 mL of seed medium was placed in a 1L Erlenmeyer flask, sterilized by moist heat at 121°C for 30 min, and cooled to room temperature. Take a strain of pET9a-GCSF / BL21(DE3), inoculate it in the seed medium, the inoculation amount is 0.03%, the culture temperature is 30°C, the speed of the shaker is 250rpm, and the OD is measured for 8h. 600 As 1.92, a fermented seed culture solution was obtained. The components and contents of the seed medium are: selective soybean peptone 10g / L, yeast powder 5g / L, NaCl 5g / L, prepared with purified water, natural pH. Microscopic examination showed that there was no bacterial contamination in the seed culture solution, which was a typical form of Escherichia coli.
[0053] 2. Preparation before inoculation: First, clean the fermentation tank, dissolve the fermentation medium in purified water, stir evenly, transfer it to the fermentation tank, st...
Embodiment 2
[0059] Embodiment two (take 5L fermentation tank as an example)
[0060] 1. Cultivation of seeds: 200 mL of seed medium was placed in a 1L Erlenmeyer flask, sterilized by moist heat at 121°C for 30 min, and cooled to room temperature. Take a strain of pET9a-GCSF / BL21(DE3), inoculate it in the seed medium, the inoculation amount is 0.03%, the culture temperature is 30 ℃, the shaking speed is 250 rpm, and the OD is determined by shaking culture for 9 hours. 600 As 2.20, a fermented seed culture solution was obtained. The components and contents of the seed medium are: selective soybean peptone 10g / L, yeast powder 5g / L, NaCl 5g / L, prepared with purified water, natural pH. Microscopic examination showed that there was no bacterial contamination in the seed culture solution, which was a typical Escherichia coli form.
[0061]2. Preparation before inoculation: First, clean the fermentation tank, dissolve the fermentation medium in purified water, stir evenly, transfer it to the fe...
Embodiment 3
[0066] Embodiment three (take 5L fermentation tank as an example)
[0067] 1. Cultivation of seeds: 200 mL of seed medium was placed in a 1L Erlenmeyer flask, sterilized by moist heat at 121°C for 30 min, and cooled to room temperature. Take a strain of pET9a-GCSF / BL21(DE3), inoculate it in the seed medium, the inoculation amount is 0.03%, the culture temperature is 30 ℃, the speed of the shaking table is 250 rpm, and the OD is determined by shaking culture for 10 hours. 600 As 2.47, a fermented seed culture broth was obtained. The components and contents of the seed medium are: selective soybean peptone 10g / L, yeast powder 5g / L, NaCl 5g / L, prepared with purified water, natural pH. Microscopic examination showed that there was no bacterial contamination in the seed culture solution, which was a typical Escherichia coli form.
[0068] 2. Preparation before inoculation: First, clean the fermentation tank, dissolve the fermentation medium in purified water, stir evenly, transfe...
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