Large-scale serum-free culture method for rhIL-12 engineering 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 biological technology, and relates to a cell culture medium and a culture method. Specifically, the present invention is a method for large-scale cultivation of recombinant human interleukin-12 engineered cells using a serum-free and protein-free medium. By using this method to cultivate recombinant human interleukin-12 engineered cells, high Yield, high activity of recombinant human interleukin-12. Background technique [0002] Human Interleukin-12 (Human Interleukin 12, hIL-12) is a heterodimeric cytokine composed of two subunits p35 and p40 linked by multiple pairs of disulfide bonds. hIL-12, also known as NK cell stimulating factor (NKSF), has strong anti-tumor and anti-viral effects. This effect is mediated by the body's natural killer cells (Natural killer cells, NK cells). hIL-12 also It plays an important role in the development, differentiation, maturation and activation of cytotoxic T cells, helper T ce...

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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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