牛白细胞介素-2在毕赤酵母中的表达(英文)

Expression of Bovine Interleukin-2 Gene in Pichia pastoris

将编码牛白细胞介素 - 2 (Bo IL2 )成熟肽的 c DNA克隆到巴斯德毕赤酵母 (Pichia pastoris)表达载体 p PICZB中 ,构建出含 Bo IL 2基因的重组质粒 Bo IL 2 - p PICZB。将经 Sac 酶切后线性化的 Bo IL 2 - p PICZB电转化到巴斯德毕赤酵母X- 33中 ,转化子经高浓度 Zeocin抗性筛选鉴定后 ,用 1 %甲醇诱导目的蛋白表达。经 SDS- PAGE及 Western blotting检测 ,表明 Bo IL2在酵母中获得了胞内表达 ;通过金属螯合亲和层析 (MCAC)获得纯化的重组蛋白 ;培养小鼠CTL L 2细胞进行活性检测 ,证实所表达的重组 Bo IL

The Interleukin-2 gene cDNA was cloned into the Pichia pastoris expression vector pPICZB,which is under the control of the alcohol oxidase promoter AOX1.The linearized recombinant plasmid of BoIL2-pPICZB,digested by SacⅠ,was transformed into X-33 strains by electroporation.The multi-copy insert transformants were screened by Zeocin-resistance and induced by 1% methanol.The intracellular expression products were tested by SDS-PAGE analysis and Western blotting.Purified recombinant BoIL2 was gained by metal-chelating affinity chromatographic (MCAC).Assay with murine CTLL-2 cells showed that the recombinant BoIL2 exhibited the biological activity.