阿达木单抗抗原结合片段在大肠杆菌中的共分泌表达和纯化

Co-Expression of Adalimumab Antigene-Binding Fragments in E. coli and Purification

构建阿达木单抗抗原结合片段(Fragment antigen binding,Fab)表达载体,使其在大肠杆菌内高效共分泌表达,周质空间中获得具有活性的目的蛋白。将带有信号肽的Fab片段重链与轻链克隆于带有双启动子的pETDuet-1载体内,BL21(DE3)Star宿主菌内利用IPTG低温诱导双基因共分泌表达,选用"渗透压冲击法"提取周质蛋白,色谱柱纯化,Western blot分析纯化结果,与纯化的人肿瘤坏死因子(TNF-α)相互作用进行活性分析。SDS-PAGE与Western blot分析在细胞周质中同时存在重链和轻链,大小与预期相符,ELISA试验显示Fab片段与TNF-α结合。结论:得到有活性的人源的阿达木单抗的Fab的可溶性表达产物,为进一步对其进行结构与功能的研究奠定了基础。

To obtain the active antigen-binding fragment( Fab) of Adalimumab from E. coli by constructing a secretory expression system using'double gene'expression vector,the cDNA of light chain and heavy chain of Adalimumab fab along with signal peptide PelB and OmpA respectively,were cloned into vector pETDuet-1. The resulting plasmids was transformed in BL21( DE3) Star,induced by IPTG at low temperature. The active Adalimumab Fab protein was obtained through the periplasmic extraction,affinity purification,ion exchange and size exclusion chromatography. The Adalimumab Fab protein was analyzed by SDS-PAGE,Western blot and ELISA. SDS-PAGE and Western Blot analysis showed that both heavy chain and light chain were successfully translocated into the periplasm of E. coli and the formed heterologous dimer. The purified Adalimumab Fab protein from multipul purification steps could interact with its antigen TNF-α. Active Adalimumab Fab protein was successfully obtained from E. coli periplasm using'double gene'expression vector,which provided an economic and convenient method for the study on biological function and industry production of Adalimumab Fab.