High density fermentation method of recombinant porcine interferon alpha 1 (rPoIFN alpha 1) gene engineering bacteria

A genetically engineered bacteria and high-density fermentation technology, applied in the field of fermentation process, can solve the problems of difficulty in meeting industrialization requirements, increasing renaturation steps, and low titer, avoiding the need for renaturation problems, efficient expression, shortening The effect of the production cycle

Active Publication Date: 2014-02-19
王明丽 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, the large-scale fermentation production technology of genetically engineered recombinant porcine interferon α has the following problems: 1) Most of the expressed porcine interferon α protein exists in the form of inactive inclusion bodies,

Method used

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  • High density fermentation method of recombinant porcine interferon alpha 1 (rPoIFN alpha 1) gene engineering bacteria
  • High density fermentation method of recombinant porcine interferon alpha 1 (rPoIFN alpha 1) gene engineering bacteria
  • High density fermentation method of recombinant porcine interferon alpha 1 (rPoIFN alpha 1) gene engineering bacteria

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Embodiment 1

[0032] (1) Seed fungus cultivation: inoculate the seed culture with the strains of the working seed fungus stored at --- 20°C, and use a rotary constant temperature and speed-adjustable shaker for cultivation; control the rotation speed at 220r / min, cultivate the temperature at 37°C, and the initial pH Value 7.1, culture time 10h;

[0033] (2) Fermentation: Put the cultured seed liquid into a 30L fermenter according to the inoculation amount of 1% for fermentation (the amount of fermentation medium is 20L), that is, pass sterile air into the fermenter with a ventilation rate of 1.5L / min to maintain The oxygen content in the fermentation tank is 10-20%, the culture temperature is set at 37°C, the mechanical stirring speed is 220r / min, and 3mol / L ammonia water is added to control the fermentation pH at 7.0-7.1;

[0034] (3) Feed: ferment for 2 to 3 hours, measure OD with UV spectrophotometer 600 After it is 0.4~1.0, add peptone 20~100g, yeast powder 10~40g, glucose 5~20g every ...

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Abstract

The invention discloses a high density fermentation method of recombinant porcine interferon alpha 1(rPoIFN alpha 1) gene engineering bacteria, and belongs to the technical field of fermentation engineering. The high density fermentation method of the recombinant porcine interferon alpha 1 (rPoIFN alpha 1) gene engineering bacteria is as follows: recombinant escherichia coli BL21/pET-32a-rPoIFN alpha 1 which can construct and express rPoIFN alpha 1 by itself is used as a production bacteria strain for fermentation at 37 DEG C for 2-3h, inducer isopropyl-beta-d-thiogalactoside (IPTG) with the concentration of 50 g / L-100 g / L is added into each liter fermentation solution by one time, fermentation is continued at 32 DEG C for 4-5 h, and then the fermentation process is completed. The high density fermentation method can realize the high-efficiency expression of the rPoIFN alpha 1, an objective protein expression amount accounts for more than 40% of the total bacteria protein, the potency can reach above 1*10<6>IU/ml, the protein is a soluble protein, denaturation and renaturation problems of inclusion body expression can be avoided, the production cycle is shortened, and the high density fermentation method provides a solid foundation for industrial production of the rPoIFN alpha 1.

Description

technical field [0001] The invention mainly relates to a high-density fermentation method of recombinant porcine interferon α1 genetically engineered bacteria, which belongs to the technical field of fermentation process. Background technique [0002] Interferon (IFN) is a protein with broad-spectrum anti-virus, anti-tumor and immunoregulatory effects induced by viral infection. It mainly inhibits the growth, reproduction and development of viruses by inhibiting viral gene transcription and degrading viral RNA. Antitumor activity. Interferon is divided into three types: α, β, and γ according to the different production cells, biochemical characteristics, and the role it plays in the body's immunity. The mechanism by which α-interferon can exert broad-spectrum and high-efficiency antiviral effects in vivo and in vitro is mainly to inhibit the synthesis of viral proteins, and can selectively act on infected cells without any drug residue after use. At present, more than ...

Claims

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Application Information

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IPC IPC(8): C12P21/02
Inventor 王明丽赵俊陈琨
Owner 王明丽
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