人源抗HBsAg单链抗体在巴氏毕赤酵母中的表达

在P.pastoris中分泌表达非融合抗HBsAg单链抗体（HBscFv）。设计引物从pGEMHBscFv上扩增目的基因，亚克隆至P.pastoris表达载体pPICZαA中，线性化后转化P. pastorisGS115；转化子经菌落PCR、高浓度Zeocin抗性筛选鉴定后，甲醇诱导目的蛋白表达。结果发现，重组HBscFv可以在α因子的引导下，分泌至培养基中，产量为80mg/L；分泌至培养基中的HBscFv具有结合HBsAg活性，活性总量在诱导培养72h后达最高峰，在诱导培养后期，HBscFv活性下降；PAS糖显色结果表明，酵母表达HBscFv是一种低糖基化蛋白或非糖基化蛋白。

Expression of a Human Single-chain Fv Antibody Against HBsAg in Pichia pastoris

To express and secrete native HBscFv (anti-HBsAg single-chain Fv) in P. pastoris, HBscFv was amplified from plasmid pGEM-HBscFv, and then sub-cloned into expression vector pPICZalphaA. The resulting plasmid pPIC-HBscFv was linearized and transformed into P. pastoris GS115. The recombinant Pichia strains, identified by direct PCR and Zeocin-resistant screening of Pichia transformants, were cultured and induced with methanol. It was found that recombinant HBscFv, lead by alpha-factor, could be secreted into the culture supernatant to a level of 80mg/L. The bioactivity of Pichia produced HBscFv was confirmed by indirect ELISA, which also suggested that the bioactivity of HBscFv in the culture supernatant reached its peak in 72h and decreased in the late-stage of the induction. PAS staining suggests that HBscFv produced by yeast is poorly glycosylated or none-glycosylated protein.