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Fermentation method of engineering bacteria for preparing recombination human granular cell colony stimulation factor

A colony-stimulating factor and fermentation method technology, which is applied in the field of preparation of medicinal protein substances, can solve the problems of low activity, low expression rate, and low cell yield

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 activity and high cost

Method used

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Embodiment Construction

[0015] The present invention will be described in further detail below.

[0016] The invention relates to a fermentation method of an engineered bacteria for preparing recombinant human granulocyte colony stimulating factor. The engineered bacteria is DH5α-PBV220-hGCSF. The fermentation process includes seed liquid culture and batch fed fermentation. The seed liquid culture Including the following sub-steps: ①. Screening of strains: remove the engineered strains from the strain storage library, cultivate them overnight at 30°C on LB plates, pick a single colony and inoculate it in a 5ml LB test tube containing 100μg / ml ampicillin for cultivation To OD 600 The value is 0.4-0.6, and then cultured at 42°C for 4 hours. The cells are collected by centrifugation and analyzed by SDS-PAGE. The clone with the highest expression of recombinant human granulocyte colony stimulating factor is used as the seed for fermentation. Take one; ②. First-level seed solution culture: inoculate the seeds...

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Abstract

A fermenting method for preparing the engineering bacterium DH5 alpha-PBV220-hGCSF used to prepare the recombinant human granulocyte colony stimulating factor includes such steps as screening bacterial strain, culturing in the class-one seed liquid, culturing in class-two seed liquid, inoculating basic fermenting and induced fermenting while supplementing material every one hour when the OD600 is 2, and terminating the fermentation. Its advantages are high output and high activity.

Description

Technical field [0001] The invention relates to the preparation of medicinal protein materials, in particular to a method for fermenting genetically recombined engineered bacteria used for preparing target proteins, and in particular to a method for fermentation of engineered bacteria for preparing 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 and Souza from AMGEN in the United States .LM further determined the N-segment amino acid sequence of this hG-CSF, cloned the hG-CSF gene from the 5637 cell line, and inserted the gene into E. coli using genetic engineering technology to successfully prepare hG-CSF and developed Recombinant human granulocyte colony stimulating factor (hereinafter referred to as rhG-CSF), the trade name is Filgratin (Wheer blood). ...

Claims

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Application Information

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IPC IPC(8): C12P21/02C12N1/20
Inventor 李政海李静李继东
Owner 深圳未名新鹏生物医药有限公司
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