CAJ | 학술논문

将编码牛白细胞介素-2(BoIL2)成熟肽的cDNA克隆到巴斯德毕赤酵母(Pichia pastoris)表达载体pPICZB中,构建出含BoIL2基因的重组质粒BoIL2-pPICZB.将经Sac Ⅰ酶切后线性化的BoIL2-pPICZB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BoIL2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CTLL2细胞进行活性检测,证实所表达的重组BoIL2具有生物活性.
장편마우백세포개소-2(BoIL2)성숙태적cDNA극륭도파사덕필적효모(Pichia pastoris)표체재체pPICZB중,구건출함BoIL2기인적중조질립BoIL2-pPICZB.장경Sac Ⅰ매절후선성화적BoIL2-pPICZB전전화도파사덕필적효모X-33중,전화자경고농도Zeocin항성사선감정후,용1%갑순유도목적단백표체.경SDS-PAGE급Western blotting검측,표명BoIL2재효모중획득료포내표체;통과금속오합친화층석(MCAC)획득순화적중조단백;배양소서CTLL2세포진행활성검측,증실소표체적중조BoIL2구유생물활성.
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 Sac I ,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.