A large-scale serum-free culture method of rhil-12 engineered cells

Abstract

The invention relates to a large-scale serum-free culture method for rhIL-12 engineering cells, which comprises the following step of: inoculating rhIL-12 engineering cells in a logarithmic phase into a serum-free and protein-free medium for culture, wherein the medium is a CD CHO liquid medium comprising sodium pyruvate at the final concentration of 0.8 to 1.2mM, hypoxanthine at the final concentration of 0.075 to 0.125mM, thymidine at the final concentration of 0.012 to 0.020mM, adenosine at the final concentration of 0.5 to 0.9mg / L, guanosine at the final concentration of 0.5 to 0.9mg / L, cytidine at the final concentration of 0.5 to 0.9mg / L, uridine at the final concentration of 0.5 to 0.9mg / L, L-glutamine at the final concentration of 0.4 to 0.8mg / L, L-asparagine at the final concentration of 0.4 to 0.8mg / L, L-proline at the final concentration of 1.5 to 2.0mg / L and non-essential amino acid at the final concentration of 0.08 to 0.125mM. The invention also provides a medium used in the method. By the medium and the culture method, a high-yield and high-activity recombinant human interleukin-12 can be obtained.

Description

Technical field [0001] The invention belongs to the field of biotechnology and relates to cell culture media and culture methods. Specifically, the present invention is a method for large-scale culturing of recombinant human interleukin-12 engineered cells using a serum- and protein-free medium. The method is used to cultivate recombinant human interleukin-12 engineered cells to obtain high Yield, highly active recombinant human interleukin-12. Background technique [0002] Human Interleukin 12 (hIL-12) is a heterodimeric cytokine composed of two subunits of p35 and p40 connected by multiple pairs of disulfide bonds. hIL-12, also known as NK cell stimulating factor (NKSF), has strong anti-tumor and anti-viral benefits. This effect is mediated by the body’s natural killer cells (NK cells). hIL-12 also It plays an important role in the development, differentiation and maturation of cytotoxic T cells, helper T cells, and B cells. It is known as the core regulatory factor of the im...

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Problems solved by technology

Before the present invention, some researchers respectively inserted the genes of p40 and p35 into the same vector for co-transfection (Dai Yan, Chen Weifeng. Expression of human recombinant interleukin 12 in CHO cells. Biochemistry and Biophysics Progress, 1998, 25(3): 254-258), or the gene fusion of p40 and p35 to construct IL-12 single-chain eukaryotic expression vector for expression (Zhang Mei, Si Lusheng, Jin Li et al. Recombinant human single-chain Construction of interleukin-12 fusion gene, eukaryotic expression and biological activity determination. Chinese Journal of Hematology, 2000, 21(12): 636-640), or conventional culture of vector cells transfected with hIL-12 (Liu Meng, Zheng Shanshan. Recombinant human interleukin 12 gene in CHO-dhfr - Stable expression of cells. Journal of Immunology, 1996, 12(4): 215-219), but failed to achieve high expression of rhIL-12 in large-scale serum-free culture of carrier cells

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