重组人肿瘤坏死因子-α(rhTNFα)工程菌高密度发酵研究

Study on High Cell-Density Fermentation of Recombinant E.coli Expressing rhTNFα

报道了 rh TNFα重组菌发酵条件及高密度、高表达发酵的研究。首先研究了营养成份对其生长和表达的影响 ,确定了半合成发酵培养基 ;筛选出了 rh TNFα生长和表达的最适宿主菌 E.coli YK5 37;研究了影响高表达的几个关键因素 ,建立了该类型重组菌的诱导表达条件。结果表明限制发酵过程中乙酸积累、控制在指数生长期进行诱导、持续诱导时间不超过 5 h是实现高密度、高表达发酵的关键条件。采用半经验补料流加公式 ,在 NBS Bio Flo30 0 0型 5 L 发酵罐中利用恒溶氧 -补料培养批技术 ,限制甘油浓度在 5 g/ L,可使 E.coli YK5 37/ p SB- TR最终菌体密度和 rh TNFα的产量分别达到 A6 0 0 12 6和 8.4g/ L。

Semi-defined medium was established and the most suitable host E.coli YK537 was selected through the study of effects of nutrients on cell growth and rhTNFα expression. The main factors affecting the rhTNFα expression level were optimized. Results indicated that keeping acetic acid concentration at low level, inducing at log phase and maintaining induction for less than 5 h were critical to high cell-density and high expression level in fermentation. The DO-stat fed-batch fermentation for E.coli YK537/pSB-TR was carried out in NBS BioFlo 3000 5 L fermentor with glycerol concentration of 5g/L control using medium addition formula, and the final cell density and the yield of rhTNFα achieved A 600 126 and 8.4 g/L, respectively.\;