Method for producing recombinant human granulocyte colony stimulating factor

A technology of colony-stimulating factor and production method, which is applied in the biological field and can solve the problems of low renaturation rate and high production cost of recombinant human granulocyte colony-stimulating factor

Active Publication Date: 2006-08-09
山东泉港药业有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to overcome the defects of low renaturation rate and high production cost of recombinant human granulocyte colony-stimulating fac

Method used

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  • Method for producing recombinant human granulocyte colony stimulating factor
  • Method for producing recombinant human granulocyte colony stimulating factor
  • Method for producing recombinant human granulocyte colony stimulating factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Fermentation: Inoculate the engineered strains on the LA plate, cultivate them at 30°C, and after they grow into colonies, pick a single colony and inoculate it in 10ml LA liquid culture medium, shake it at 30°C to obtain a first-grade seed liquid, and transfer it to 1% in LA liquid medium, shaking culture to obtain secondary seed liquid.

[0028] Prepare the fermentation medium, put the part that can withstand high pressure into the fermenter for high-pressure sterilization, filter and sterilize the components that cannot withstand high pressure, add it after the liquid in the fermenter cools down, and add the newly cultivated seed liquid at 10% Proportional inoculation in fermenter for fermentation. Control the culture temperature at 30°C, adjust the pH value with ammonia water to stabilize the pH between 6.8 and 7.4, control the dissolved oxygen > 70% by adjusting the ventilation volume and rotation speed, and feed appropriately. Enrichment to OD 600 At 2.5-3.0, th...

Embodiment 2

[0030] Bacteria destruction and inclusion body washing: suspend the wet bacteria with TE (50mmol / LTris-Cl, 5mmol / LEDTA) at a ratio of 1:10 (g / ml), add lysozyme to a final concentration of 1mg / ml, 4 Place at ℃ for 4 hours, and ultrasonically break in an ice bath, 6 minutes each time, with an interval of 3 minutes, a total of 7 times. Centrifuge at 4°C and 8500 rpm for 15 min, discard the supernatant, resuspend the pellet in TE buffer, repeat the above process twice, and wash the pellet twice with 2 mol / L urea.

[0031] The precipitate was dissolved with 8mmol / L urea, 2mmol / L EDTA, 1mmol / L DTT, 50mmol / L Tris-HCl pH 8.0, centrifuged at 8500rpm at 4°C for 15min, and the supernatant was the inclusion body eluate.

Embodiment 3

[0033] Molecular sieve chromatography: take the above-mentioned inclusion body dissolution solution, add DTT to a final concentration of 5mmol / L, put it on the column in a water bath at 30°C for 30min, and the mobile phase is 8mmol / L urea, 2mmol / L EDTA, 1mmol / L LDTT, 50mmol / L Tris-HCl pH 8.0, the flow rate is about 0.7ml / min, and the elution peaks are collected in sections. The chromatographic curve is shown in Figure 2. The target protein is located in the second peak, and the impurity protein with a molecular weight greater than 30KD can be removed by molecular sieve purification, as shown in Figure 3.

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Abstract

This invention relates to a method for producing recombined human granular leukocyte colony stimulating factors including: fermenting, breaking bactrriums, extracting occlusion bodies, chromatographing with molecular sieves, renaturating, exchanging anions, exchanging cations to get the raw fluid, in which, BL21 is selected as the host bacterium and the engineering bacterium type is got in high expression volume, quick reproduction and stable passage, several purification steps greatly reduce the residural toxin in the bacterium and the specific activity is increased greatly, a dialysis method is applied for the renaturation to reduce the concentration of the denaturalization agent steadily so the renaturation rate is at high level.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a production method of recombinant human granulocyte colony-stimulating factor. Background technique [0002] Granulocyte colony-stimulating factor (G-CSF) can promote the proliferation and differentiation of granulocyte hematopoietic stem cells and enhance the function of mature granulocytes. Agranulocytosis, and neutropenia associated with aplastic anemia have obvious curative effect. [0003] The biological activity of genetically engineered recombinant G-CSF is similar to that of natural one, and it can be produced in large scale to meet clinical needs. In the existing production method, Escherichia coli expresses foreign proteins, most of which exist in the form of insoluble inclusion bodies, which do not have biological activity and need to be denatured and refolded during the production process to restore their biological activity. The ubiquitous problem is the low renaturat...

Claims

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Application Information

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IPC IPC(8): C12P21/02C07K14/52C07K1/18
Inventor 陈文芳宋磊刘红军
Owner 山东泉港药业有限公司
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