制备可溶性肿瘤坏死因子受体Ⅱ-脂联素球部融合蛋白的两种细胞培养工艺比较  

Comparison of two types of cell cultures for preparation of sTNFRII-gAD fusion protein

作　　者：黄世高[1] 尹玉婷[1] 熊春晖[1] 王彩虹[1] 吕建新[1] 高基民[1] 

机构地区：[1]温州医学院浙江省模式生物技术与应用重点实验室,浙江温州325035

出　　处：《生物工程学报》2013年第1期115-118,共4页Chinese Journal of Biotechnology

基　　金：国家新药创新重大专项(No.2009ZX09103-649);浙江省重大科技专项(Nos.2008C14082;2010C13007);卫生部科研基金(No.201231029);浙江省教育厅科学基金(No.Y201016528);浙江省卫生高层次创新人才培养工程;浙江省研究生创新科研项目资助~~

摘　　要：利用7．5L生物反应器篮式贴壁培养和全悬浮批式培养CHO工程细胞株表达可溶性肿瘤坏死因子受体Ⅱ-脂联素球部（sTNFRII-gAD）融合蛋白，比较这两种培养方法的产率，以便优化高效表达sTNFRII—gAD融合蛋白的制备工艺。篮式贴壁培养首先小规模培养CHO工程细胞株，待细胞增殖到一定密度后以3×105-4×10^5cells／mL密度接种生物反应器贴壁培养3d，调换成不含血清的LK021培养基继续培养4d。而全悬浮无血清批式培养则以3×10^5-4×10^5cells／mL密度的CHO工程细胞株接种于生物反应器，连续培养7d。培养过程实时监测培养条件，维持pH和DO的稳定。分别收集细胞上清，离心去细胞后用Pellicon切相流超滤系统对蛋白进行浓缩，并通过DEAE离子交换柱进行纯化。结果显示，篮式贴壁培养和全悬浮批式培养均成功表达了sTNFRII-gAD融合蛋白，产量分别为8．0mg／L和7．5mg／L、纯度分别为95％和98％，从而为sTNFRII-gAD融合蛋白的中试工艺研究提供了一定的基础。In this study we used two types of cell cultures, i.e., anchorage-dependent basket and full cultures of sTNFRll-gAD-expressing CHO cells in the CelliGen 310 bioreactor （7.5 L） to compare their optimize the culturing conditions for efficient expression of sTNFRII-gAD fusion protein consisting necrosis factor receptor Ⅱ and globular domain of adiponectin. The anchorage-dependent basket culture suspension batch yields in order to of soluble tumor was performed in 4L 10% serum-containing medium with the final inoculating concentration of 3×105 to 4×105 cells/mL of sTNFRII-gAD-expressing CHO cells for 3 days, and then switched to 4 L serum-free LK021 medium to continue the culture for 4 days. The full suspension batch culture was carried out in the 4 L serum-free LK021 medium with the final inoculating concentration of 3×105 to 4×105 cells/mL of sTNFRII-gAD-expressing CHO cells for 7 days. The culturing conditions were monitored in real-time to maintain pH and dissolved oxygen stability through the whole process. The supernatants were collected by centrifuge, and the protein was concentrated through Pellicon flow ultrafiltration system and then purified by DEAE anion exchange. The results showed that the yields of sTNFRII-gAD fusion protein were 8.0 mg/L with 95% purity and 7.5 mg/L with 98% purity in the anchorage-dependent basket and the full suspension batch cultures, respectively. The study provided the framework for the pilot production of sTNFRII-gAD fusion protein.

关 键 词：可溶性肿瘤坏死因子受体Ⅱ-脂联素球部融合蛋白 生物反应器 CHO细胞 真核表达 

分 类 号：R730.5[医药卫生—肿瘤]