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目的:用生物发酵方法大量制备含重组质粒pBAD/HBs-Fab-IFN-α的Topl0大肠杆菌,用金属亲和层析方法纯化大肠杆菌表达产物,获得大量高纯度的人源抗HBS-Fab-IFN-α融合蛋白。方法:选取含重组质粒pBAD/HBs-Fab-IFN-α的Topl0大肠杆菌单克隆菌落,通过制备一级及二级种液,按比例加入5L发酵罐中进行发酵,发酵过程中在菌体起始密度、诱导剂加入时机、诱导时间以及诱导温度等条件探索成熟的基础上,采用50L大型发酵罐进行批量发酵。所获菌体蛋白通过饱和硫酸铵粗提和Ni-NTA金属螯和层析获得较纯的表达产物,比较表达量,鉴定纯化后蛋白的抗原活性及生物学活性。结果:用5L及50L发酵罐能获得较高蛋白产量的大肠杆菌,经饱和硫酸铵粗提及Ni-NTA金属螯和层析纯化后可获得抗原性及生物学活性较好的抗体片段。结论:发酵罐发酵大肠杆菌,产量高,所获蛋白含量活性较稳定,为批量生产作了准备。
OBJECTIVE: To prepare a large amount of Top 10 Escherichia coli containing recombinant plasmid pBAD / HBs-Fab-IFN-α by bio-fermentation and to purify Escherichia coli expression product by metal affinity chromatography to obtain a large amount of high purity human anti-HBS-Fab- IFN-α fusion protein. Methods: Monoclonal colonies of Top10 E.coli containing recombinant plasmid pBAD / HBs-Fab-IFN-α were selected and fermented by adding 5L fermentor in proportion to primary and secondary seed solutions. During fermentation, Initial density, inducing agent adding timing, induction time and induction temperature were explored on the basis of maturity, using 50L large fermentor for batch fermentation. The obtained bacterial protein was purified by saturated ammonium sulfate and Ni-NTA metal chelation chromatography to obtain the pure expression product. The expression level was compared and the purified protein’s antigenic activity and biological activity were identified. Results: Escherichia coli with high protein yield could be obtained with 5L and 50L fermenters. After purification by saturated ammonium sulphate and Ni-NTA metal chelation chromatography, better antigenic and biologically active antibody fragments were obtained. Conclusion: Fermentor can ferment Escherichia coli with high yield and stable protein content, which is ready for mass production.