Preparation method of recombination human granular cell colony stimulating factor

A technology of colony-stimulating factor and granulocytes, applied in the field of preparation of pharmaceutical protein substances, can solve the problems of low renaturation rate, low expression rate, and low activity of inclusion bodies

Active Publication Date: 2006-01-11
深圳未名新鹏生物医药有限公司
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Problems solved by technology

[0009] The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art, and start with high-density and rapid cultivation of recombinant genetically engineered bacterial strains to solve the problems of low expression rate and low bacterial cell yield in the preparation of rhG-CSF , low inclusion body renaturation rate, low activity and high cost
[0011] Compared with the prior art, the preparation method of recombinant human granulocyte colony-stimulating factor of the present invention provides a solution to the problems of low inclusion body renaturation rate, low activity and high cost in the preparation of rhG-CSF. possible

Method used

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Embodiment Construction

[0012] The present invention will be further described in detail below.

[0013] The preparation method of the recombinant human granulocyte colony-stimulating factor of the present invention comprises the steps of: a. Selecting the engineering bacteria DH5α-PBV220-hGCSF strain for fermentation and culture; b. Separating and washing the inclusion bodies of the bacteria obtained from the fermentation culture to obtain refined Inclusion bodies; c. Denaturing the refined inclusion bodies to obtain a denatured product; d. Carrying out the first renaturation treatment on the denatured product to obtain the first renaturation product; e. The first renatured product Carry out ultrafiltration treatment on the product to obtain the ultrafiltration product; f. carry out the second renaturation treatment on the ultrafiltration product to obtain the second renaturation product; g. carry out chromatographic treatment on the second renaturation product , to obtain recombinant human granuloc...

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Abstract

A process for preparing the recombinant human granulocyte colony stimulating factor includes such steps as fermenting culture of DH5 alpha-PBV220-hGCSF strain, separating cytoryctes, washing, modifying renaturation, ultrafilter, the second renaturation, and chromatography. Its advantages are high purity and high activity.

Description

technical field [0001] The invention relates to the preparation of medicinal protein substances, in particular to a method for preparing target proteins by using gene recombination engineering bacteria, in particular to a preparation method for recombinant human granulocyte colony-stimulating factor. Background technique [0002] In 1985, Welte.K successfully purified and refined human granulocyte colony-stimulating factor (hG-CSF) from the culture supernatant of human bladder cancer cell line 5637, and then Welte.K cooperated with Souza of AMGEN Company in the United States. .L.M further determined the N segment amino acid sequence of this hG-CSF, cloned the hG-CSF gene derived from 5637 cell line, and used genetic engineering technology to insert the gene into Escherichia coli to successfully produce hG-CSF and develop it Recombinant human granulocyte colony-stimulating factor (hereinafter referred to as: rhG-CSF), trade name Filgratin (Wheel blood). The drug was approved...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C12N1/20C07K14/525C07K1/34
Inventor 李政海胡家华李继东
Owner 深圳未名新鹏生物医药有限公司
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