猪带绦虫TSO18基因真核表达载体的构建及其表达

Construction and expression of recombinant antigen TSO18 gene of Taenia solium

采用PCR从重组克隆载体pGEMTSO18中扩增出猪带绦虫六钩蚴TSO18基因,与毕赤酵母分泌性表达载体pPIC9k相连接,构建重组表达载体pPIC9kTSO18,转化大肠埃希氏菌JM109,经测序证实,基因序列完全正确。制备重组质粒pPIC9kTSO18,用SalⅠ线性化,并电转化毕赤酵母(Pichiapastoris)GS115,使重组表达载体与酵母染色体发生同源整合。采用G418抗性梯度法筛选得到高拷贝重组菌株,以甲醇进行诱导表达,SDSPAGE和Westernblotting分析结果表明,诱导表达的培养上清中表达出具有反应活性的16ku重组蛋白,目的蛋白约占培养上清液中蛋白总量的80%以上,诱导72h目的蛋白表达量为0.5mg/mL。

The TSO18 gene was subcloned into the Pichia pastoris expression vector pPIC9k and the resultant recombinant plasmid pPIC9k-TSO18 was transformed into P.pastoris GS115 by electroporation. The multi-copy recombinant P.pastoris strains were screened by G418 and induced by methanol. The expressed product was analyzed by SDS-PAGE and Western-blotting tests. The results indicated that the aim protein was expressed in P.pastoris, and had immunological activity. The expression level of aim protein was up to 0.5mg/mL.