人源抗HBS-Fab-IFN-α融合蛋白制备和纯化

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。

Ferment and purification of human anti-HBs Fab-IFN-α fuse protein

Objective: to product massive human anti-HBs fab- IFN-α by fermenting pBAD/ g IIIA Top 10 E.coli and to obtain high pure fab- IFN-α by Ni-NTA affinity chromatography. Method: E.coli Top 10 containing recombinant PBAD/HBs-fab- IFN-α were grown in LB medium from flat to spinner flask ,and then were joined into 5L fermenter. Based on mature conditions such as origination OD600 of E.coli、the time、opportunity and temperature of induction, to fermenting E.coli by 50L fermenter. Method of purifying E.coli is saturation ammonium sulphate and Ni-NTA affinity chromatography. After purification Fab- IFN-α,the antigenicity and hallmark will be verified. Result; both 5L and 50L fermenters were applicable to product massive E.coli,and antibody can keep good antigenicity and biology after purification . Conclusion: large production and high activity antibody segment was obtained by ferment and purification. The strategy is available in gene engineering technology.