摘要:
构建了人肿瘤坏死因子受体II胞外区、人脂联素球部与人IgG1 Fc的融合基因sTNFRII-gAD-Fc的真核表达载体，在CHO-S细胞中快速高效表达，探索了无血清悬浮流加培养工艺。应用重组PCR将人IgG1 Fc基因片段与sTNFRII-gAD基因片段融合，构建pMH3-sTNFRII-gAD-Fc表达载体，电转染至CHO-S细胞，挑选G418抗性的高表达单克隆，斑点杂交半定量分析和Western blot分析sTNFRII-gAD-Fc的表达，Protein A-Agarose纯化，以及拮抗TNFα的生物活性测定。最后在摇瓶、转瓶及生物反应器中进行了逐级放大的无血清悬浮批次及流加培养，即：2.0 × 106 cells/mL的接种密度，当达到4.0 × 106 cells/mL以上时开始流加培养并以每日葡萄糖的残余量2 g/L左右作为流加体积的控制参数。成功构建pMH3- sTNFRII-gAD-Fc表达载体，检测到sTNFRII-gAD-Fc在CHO-S细胞培养上清中以二聚体和多聚体形式表达，获得了高表达(75 μg/mL)单克隆细胞株，且该蛋白具有显著抑制TNFα杀伤L929细胞的活性。在摇瓶中无血清批次培养和转瓶及生物反应器中流加培养时产量分别为10.0 mg/L、18.3 mg/L和20.5 mg/L。利用CHO-S细胞系统成功实现了sTNFRII-gAD-Fc 融合蛋白的快速高效表达，为建立一套高密度、高效表达该融合蛋白的悬浮流加培养中试工艺打下了良好基础。
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.