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利用7.5 L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3×105~4×105cells/mL密度接种生物反应器贴壁培养3 d,调换成不含血清的LK021培养基继续培养4 d。而全悬浮无血清批式培养则以3×105~4×105cells/mL密度的CHO工程细胞株接种于生物反应器,连续培养7 d。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8.0 mg/L和7.5 mg/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。
The soluble tumor necrosis factor receptor-II-adiponectin (sTNFRII-gAD) fusion protein was expressed in basket-cultured and suspension-fed batch culture CHO cells by 7.5 L bioreactor. In order to optimize the preparation process for the efficient expression of the sTNFRII-gAD fusion protein. Basket Affinity Culture First, CHO cell lines were cultured in small scale. After the cells had proliferated to a certain density, the cells were inoculated with 3 × 105 ~ 4 × 105cells / mL bioreactor for 3 days at a density of 3 × 105 cells / mL and switched to serum-free LK021 culture Base continue to cultivate 4 d. The whole suspension serum-free batch culture with 3 × 105 ~ 4 × 105cells / mL density of CHO engineering cell lines inoculated in the bioreactor, continuous culture for 7 days. The culture process monitors the culture conditions in real time to maintain pH and DO stability. The cell supernatants were collected, centrifuged to remove the cells, then the protein was concentrated by Pellicon phase-flow ultrafiltration and purified by DEAE ion exchange column. The results showed that the sTNFRII-gAD fusion protein was expressed successfully in both basket-type and suspension-type batch cultures with yields of 8.0 mg / L and 7.5 mg / L, respectively, with 95% and 98% purity, respectively, gAD fusion protein pilot process provides a basis for the study.