Extraction method of recombinant human interleukin-2 fermentation inclusion body

Abstract

The invention discloses an extraction method of a recombinant human interleukin-2 fermentation inclusion body. The extraction method comprises the following steps: (1) culturing an engineering strain of an escherichia coli expression recombinant interleukin-2; (2) breaking a thallus obtained by fermentation and culturing to collect an inclusion body; (3) extracting and washing the inclusion body to obtain a refined inclusion body, wherein the engineering strain is E.Coli K802(ply-4), the culturing method comprises multiplication culture and fermentation tank culture, which both adopt a yeast and peptone-containing antibiotic-free culture medium, and the thallus is broken by adopting a high-pressure homogenizer, and is degenerated to obtain a high-purity ril-2 stock solution. The extraction method disclosed by the invention is short in production period, high in production efficiency, large in production scale, and especially suitable for industrialized production; the production cost is lowered.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a method for extracting recombinant human interleukin-2 fermentation inclusion bodies. Background technique [0002] Interleukin-2 (IL-2) is an immunoregulatory factor secreted by T cells, which can not only promote the proliferation of T cells, but also enhance the activity of NK and other immunocompetent cells. , regulation and immune surveillance play an important role. In recent years, it has attracted more and more attention because of its important role in tumor treatment. Since Taniguchi et al. first cloned IL-2 cDNA in 1983 and successfully expressed IL-2 in the monkey cell line COS-7, many scholars have successfully expressed it in E. coli. Since recombinant interleukin-2 (rIL-2) has all the functions of natural IL-2, mass production of IL-2 by genetic engineering will meet the requirements of laboratory and clinical practice. [0003] The biological activity of reco...

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Problems solved by technology

[0005] However, when the applicant used the above method to replace the engineered bacteria with E.Coli K802 (ply-4) to produce interleukin-2 fermentation inclusion bodies, it was found that the expression level of ril-2 was very low, less than 25%. , resulting in very low purity of the obtained inclusion bodies, which cannot meet the requirements of large-scale production

Then the applicant adjusted the process parameters through the method of orthogonal test according to the strain characteristics of E.Coli K802 (ply-4), but the effect was very little, and the expression level of ril-2 was still low, less than 30%.

The reason for the above phenomenon is not yet clear. It may be that when the production scale is expanded to 500L, the instability factors increase during the fermentation process, and the selection of conventional technical parameters is not suitable for E.Coli K802(ply-4).