Efficient Expression of sTNFRII-gAD-Fc Fusion Protein in CHO Cell
Abstract: In order to produce the soluble TNF receptor (sTNFR) II with good neutralizing activity against TNFα, we constructed the fusion gene sTNFRII-gAD-Fc, which encoded human sTNFRII, the globular domain of adiponectin (gAD) and IgG1 Fc, and efficiently expressed it through a “GC-rich” method for mammalian gene expression in CHO-S cells, and further established the process of serum-free suspension fed-batch culture. The fusion gene of sTNFRII-gAD-Fc was obtained by recombinant PCR, and then cloned into pMH3 expression plasmid with GC-rich motif. The plasmid was electrotransfected into CHO-S cells, which were selected by G418. sTNFRII-gAD-Fc fusion protein was analyzed by dot blot and Western blot. The TNFα-antagonizing activity of sTNFRII-gAD- Fc was determined through TNFα-induced L929 cytotoxicity assay. The suspension cultures of sTNFRII-gAD-Fc-expressing CHO-S cells were performed in a step-wise manner. We assessed the expression levels of sTNFRII-gAD-Fc in batch culture in shake flaskes (500 mL), fed-batch culture in roller bottles (2 L), and the bioreactor (7.5 L) with inoculating concentration of 2 × 106 cells/ml of sTNFRII-gAD-Fc-expressing CHO-S cells, respectively. Having reached up to more than 4 × 106 cells/ml in fed-batch cultures, the cells were added semi-continuously with the feed medium to keep the glucose concentration at 2 g/L. Stable expression clones (75 μg/mL) were obtained, and sTNFRII-gAD-Fc fusion protein was expressed as dimer and polymer forms in the supernatant, and displayed very strong TNFα-neutralizing activity. The yields of sTNFRII-gAD-Fc fusion protein were about 10.0 mg/L, 18.3 mg/L and 20.5 mg/L, respectively, in 60 mL batch culture, 200 mL and 3 L fed-batch cultures. Our efficient expression of sTNFRII-gAD-Fc fusion protein by CHO-S cells had laid a good basis for the development of pilot production process in the suspension fed-batch culture.
文章引用: 王彩虹 , 蔡秦真 , 杨红枚 , 马丽莎 , 高基民 , 柳 英 (2015) sTNFRII-gAD-Fc融合蛋白在CHO细胞中的高效表达。 免疫学研究， 3， 1-11. doi: 10.12677/IS.2015.31001
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