重組人B淋巴细胞刺激因子工程菌高密度发酵.docVIP

High Cell Density Fermentation of Recombinant Human B Lymphocyte Stimulator Xue Xiao-Chang, Bao Chun-Jie, Han Wei, Qin Xin, Zhang Cun, Cao Zhu-Rong, Li Wei-Na, Zhao Yu-Hong, Zhang Yingqi Biotechnology Center of Fourth Military Abstract: In order to reach the goal of high cell density fermentation and high level expression of recombinant human B lymphocyte stimulator (E. coli DH5?/ pKKH-BLyS). Pilot test of tube culture and flask shaking culture were done to get optimized culture conditions, including pH value for culture, inducer concentration of IPTG, induction time and duration, range of dissolved oxygen and the manner of feeding. Then, we performed a three-time repeated fermentation under the established conditions. The final cell density was about 25 A600 and more than 50 g of wet bacteria per liter could be obtained. The average expression level of recombinant protein was about 40% of total bacterial proteins. The results show that the established fermentation procedure is stable, of short cycle and with high expression, which lays the basis for further purification and large-scale production of BLyS Key Words: B Lymphocyte Stimulator, Recombinant Escherichia coli, High cell density fermentation 重组人B淋巴细胞刺激因子工程菌高密度发酵 薛晓畅 包春杰 韩苇 秦鑫 张存 曹筑荣 李维娜 赵玉红 张英起 (第四军医大学药学系生物技术中心, 西安710032) 摘　要：为实现重组人B淋巴细胞刺激因子工程菌E.coli DH5?/pKKH-BLyS的高密度、高表达发酵, 首先进行了培养条件的摸索, 确定了该工程菌的最佳培养pH值、诱导剂IPTG的浓度、诱导的最佳时间段、溶氧范围以及补料的流加方式等, 根据优化条件，用5 L自控发酵罐进行三批重复发酵, 最终菌体密度均达到25 A600以上，菌体获得量均可达湿重30 g以上，目的蛋白质的表达量占菌体总蛋白的40%左右，保持并超过了该重组蛋白在试管和摇瓶中的表达量。此发酵工艺具有周期短、产量高、重复性稳定性好等特点，为下游的纯化和进一步的大规模生产奠定了基础。 关键词：B淋巴细胞刺激因子; 重组大肠杆菌; 高密度发酵 中图分类号: Q516; TQ92 　文献标识码:A 文章编号： B淋巴细胞刺激因子（B Lymphocyte Stimulator, BLyS）是1999年由瑞士和美国科学家发现的肿瘤坏死因子（Tumor Necrosis Factor, TNF）家族的一个新成员[1]。该分子含有285个氨基酸，属于II型跨膜蛋白。BLyS主要由T细胞和树突状细胞等髓系来源的细胞分泌。BLyS以膜结合及可溶性两种方式存在于机体中，两者均具有生物学活性。研究表明BLyS在B细胞的存活和功能调节方面发挥着重要作用，它可以延长B细胞存活时间、促进其增殖，从而导致B细胞增生和自身免疫狼疮样变化的出现。BLyS的过度表达是发生自身免疫性疾病如系统性红斑狼疮、多发性硬化和类风湿性关节炎等的重要因素之一[2-4]。 BLyS已经成为治疗自身免疫性疾病的有效靶点，我们构建了人BLyS疫苗表达重组载