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Method for preparing recombinant human granulocyte colony stimulating factors screened by microorganisms, and medicament composition and preparation thereof

A colony-stimulating factor and granulocyte technology, which is applied in the direction of colony-stimulating factor, drug combination, peptide preparation method, etc., can solve the problem that the mass recovery rate is less than 20%

Active Publication Date: 2013-05-22
福建亿懿兴华生物技术开发有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Recombinant hG-CSF (rhG-CSF) is usually expressed in E.coli, which exists in the form of water-insoluble inclusion bodies, so it must be dissolved with a high concentration of denaturant, so that the dissolved protein exists in an unfolded state must be refolded and then purified using multiple chromatographic steps, but the mass recovery is less than 20%

Method used

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  • Method for preparing recombinant human granulocyte colony stimulating factors screened by microorganisms, and medicament composition and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Preparation of recombinant human granulocyte colony-stimulating factor:

[0032] 1. Select engineering bacteria DH5α-PBV220-hGCSF strain for fermentation;

[0033] 2. Wash the collected cells with TE buffer for 3 times, then ultrasonically crush for 0.5-1 hour, centrifuge at low temperature, and collect inclusion bodies; then add inclusion body washing solution, mix at 4°C for 30 minutes, and centrifuge at 4°C at low temperature ; TE buffer: 20mmol / L Tris-HCl+1mmol / L EDTA, pH8.0; inclusion body washing solution: 20mmol / L Tris-HCl+1mmol / L EDTA+2mmol / L urea+0.5%Tritonx-100, pH8.0; the volume ratio of inclusion body weight to inclusion body washing solution is 1:10;

[0034] 3. Add inclusion body solution to the inclusion body, centrifuge at 4°C for 12 hours to obtain inclusion body extract; the inclusion body solution is: 20mmol / L Tris-HCl+1mmol / L EDTA+7mmol / L Guanidine hydrochloride + 20mmol / L DTT, pH8.0; the ratio of inclusion body mass to inclusion body sol...

experiment example 1

[0043] Experimental example 1: The effect of urea concentration in the eluent of SephacrylS-200 gel column on hG-GSF

[0044] Change the concentration of urea in the eluate in Example 1 (other conditions and steps are the same as in Example 1), and study its influence on the yield and specific activity of recombinant human cell colony-stimulating factor, and the results are shown in Table 1:

[0045] Table 1:

[0046]

[0047] Urea (mmol / L)

experiment example 2

[0048] Experimental example 2: Effect of pH of eluent in SephacrylS-200 gel column on hG-GSF

[0049] Change the pH in the eluate in Example 1 (other conditions and steps are the same as in Example 1), and study its influence on the yield and specific activity of recombinant human cell colony-stimulating factor, and the results are shown in Table 2:

[0050] Table 2:

[0051]

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Abstract

The invention relates to the field of bio-pharmaceuticals, and in particular relates to a method for preparing recombinant human granulocyte colony stimulating factors screened by microorganisms. The preparation method comprises the following steps of: selecting engineering bacteria of the recombinant human granulocyte colony stimulating factors to ferment and culture; ultrasonically pulverizing bacteria bodies obtained by fermentation medium; collecting inclusion bodies; washing the inclusion bodies with inclusion body washing solution and centrifuging at a low temperature; adding inclusion body dissolution liquid into the inclusion bodies, standing for 8-12 hours at 4 DEG C and centrifuging to obtain inclusion body extraction liquid; separating the extraction liquid by using a Sephacry1S-200 gel column; collecting a plurality of recombinant human granulocyte colony stimulating factor fractions; and separating by using a SephadexG25 column to obtain the recombinant human granulocyte colony stimulating factor. The invention also relates to a medicament composition and a preparation of the recombinant human granulocyte colony stimulating factors.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a method for preparing recombinant human granulocyte colony-stimulating growth factor by screening with microorganisms, its pharmaceutical composition and preparation. Background technique [0002] Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein with 174 amino acids and a molecular weight of about 20,000. It is mainly produced by endotoxin, TNF-α and IFN-γ, which can activate monocytes and macrophages. The full length of the G-CSF gene is 2.5kb, including 5 exons and 4 introns. G-CSF has 5 cysteines, two pairs of disulfide bonds are formed between Cys36 and Cys42, Cys74 and Cys64, Cys17 For unpaired cysteines, disulfide bonds are an essential factor for maintaining the biological function of G-CSF. Human and mouse G-CSF have 73% homology at the amino acid level and have crossed biological activities. G-CSF mainly acts on the proliferation, differentiation and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C07K14/53C07K1/16A61K38/19A61P7/00
Inventor 罗诚
Owner 福建亿懿兴华生物技术开发有限公司
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