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Preparation method for recombinant human granulocyte colony stimulating factor

A technology of colony-stimulating factor and granulocytes, applied in the direction of colony-stimulating factor, cytokine/lymphokine/interferon, animal/human protein, etc., can solve the problems of cumbersome operation, low yield and high equipment requirements

Inactive Publication Date: 2014-10-29
SHANGHAI SUNWAY BIOTECH
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  • Application Information

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Problems solved by technology

However, these methods have many problems such as high equipment requirements, low yield, cumbersome operation, and easy protein precipitation.
Therefore, it is a big challenge to renature rhG-CSF inclusion bodies into biologically active proteins and to increase the renaturation yield as much as possible.

Method used

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  • Preparation method for recombinant human granulocyte colony stimulating factor
  • Preparation method for recombinant human granulocyte colony stimulating factor
  • Preparation method for recombinant human granulocyte colony stimulating factor

Examples

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Embodiment

[0032] Acquisition of rhG-CSF inclusion bodies

[0033] The rhG-CSF genetically engineered bacteria (engineering strain pBVQG8 / MM294, provided by the Chinese Academy of Military Medical Sciences) was fermented in a conventional high-density manner (the engineering bacteria were fermented in a fed-batch culture method, and when the cell density reached OD value 30, The engineered bacteria were induced to ferment to express exogenous proteins; among them, the feed medium formula was: yeast extract powder 450g / L, glucose 500g / L; the fermentation medium formula was: peptone 12g / L, yeast extract 24g / L, Glycerol 4ml / L, Glucose 20g / L, Potassium dihydrogen phosphate 2.31g / L, Dipotassium hydrogen phosphate 12.54g / L, Magnesium sulfate 0.75g / L), the bacteria were collected by 8000g centrifugal force.

[0034] The collected wet bacteria were placed at -20°C and freeze-thawed once. Take 400 grams of freeze-thawed bacteria and suspend them in the lysate (20mM Tris-HCl, EDTA1mM, DTT1.5g / L, ...

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Abstract

A preparation method for recombinant human granulocyte colony stimulating factor (rhG-CSF) comprises: performing fermentation culture on rhG-CSF gene engineering bacterium, then mixing and suspending the cultured thallus in a lysate, so as to prepare an inclusion body; performing denaturation processing on the inclusion body, and performing renaturation by using column chromatography, so as to obtain an rhG-CSF renaturation liquid; and finally performing cationic chromatography and gel filtering chromatography, and collecting and obtaining recombinant human granulocyte colony stimulating factor. By using column chromatography renaturation method, operation is simplified, and the renaturation yield of recombinant human granulocyte colony stimulating factor is higher than 70%. An rhG-CSF stock solution is obtained after the renaturation liquid is subjected to cation exchange and molecular sieve chromatography. The electrophoresis purity of the stock solution reaches 99% or more, the RP-HPLC purity reaches 98% or more, and the specific activity is 6.6*10<7> IU / mg.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, in particular to a preparation method of recombinant human granulocyte colony stimulating factor (rhG-CSF). Background technique [0002] Human granulocyte colony stimulating factor (hG-CSF) belongs to the hematopoietic growth factor family. Endotoxin, TNF-a and IFN-γ can activate monocytes and macrophages to produce G-CSF. In addition, fibroblasts, endothelial cells, stellate cells and bone marrow stromal cells can also secrete G-CSF after being stimulated and activated by LPS, IL-1 or TNF-α. Certain leukemia cells as well as CHu-2 human oral cancer cells, 5637 human bladder cancer cells, MIAPa Ca-2 pancreatic cancer cells can constitutively express G-CSF. [0003] In 1986, G-CSF cDNA was cloned successfully. The full length of C-CSF gene is 2.5kb, including 5 exons and 4 introns. Humans have two different G-CSF cDNAs, which encode precursor proteins containing 207 and 204 amino ac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C07K14/53C07K1/16
Inventor 岑仡宋宏
Owner SHANGHAI SUNWAY BIOTECH
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