幽门螺杆菌多表位肽在毕赤酵母中的分泌表达

Secretory Expression of Helicobacter Pylori Multi-Epitope Peptide in Pichia Pastoris

构建分泌表达幽门螺杆菌多表位肽CTB-UE的毕赤酵母。以原核表达载体pET-22b-CTB-UE为模板,通过PCR扩增得到融合His-tag的ctb-ue基因序列,将其插入酵母表达载体pPIC9K,得到重组表达载体pPIC9K-CTB-UE。重组表达载体pPIC9K-CTB-UE经内切酶Sal I线性化后,电击转化进毕赤酵母GS115,通过组氨酸缺陷性MD平板筛选重组菌株,G418抗性筛选多拷贝转化子,阳性转化子经PCR鉴定,摇瓶发酵表达,取上清经过超滤浓缩,Ni-NTP亲和层析纯化,透析脱盐,SDS-PAGE检测,Western blot验证。结果获得了整合有ctb-ue基因的毕赤酵母,诱导后分泌表达的CTB-UE蛋白能够与His-tag抗体发生特异性反应。成功构建了分泌型毕赤酵母GS115(pPIC9K-CTB-UE),能分泌表达幽门螺杆菌多表位肽CTB-UE。

To construct Pichia pastoris secretory expression strain of Helicobacter pylori muhi-epitope peptide CTB-UE, with the plasmid pET-22b-CTB-UE as template, the gene sequence of ctb-ue with His-tag was amplified by PCR and then inserted into the P. pastoris secretory expression vector pP1C9K. Recombinant plasmid pPIC9K-CTB-UE was linearized by Sal I and transformed into yeast host strain P. pastoris GS115 by electroporation. Recombinant Transformants were selected on MD plates and then incubated on YPD plates containing different concentrations of G418 to select high G418 resistant and multicopy integrants so as to obtain highly expressed engineered strains. The positive transformants were identified by PCR and fermented in flask. The supematant from the shake flask culture was collected by centrifugalization, concentrated by ultrafiltration,purified with Ni-NTP column ,desalted by dialysis and assayed by SDS-PAGE and Western blot. It was proved by PCR that gene sequence of ctb-ue was integrated into the regions of homology within the yeast genome. After induction, CTB-UE protein was secreted continuously into the medium. Western blot analysis demonstrated that the purified CTB-UE reacts with monoclonal antibody of His-tag. P. pastoris secretory expression strain GS115 （pPICgK-CTB-UE） was successfully constructed and Helicobacter pylori muhi-epitope peptide CTB-UE was secreted continuously.