人源性抗角蛋白单链抗体在巴氏毕赤酵母的分泌表达

目的：用巴氏毕赤酵母表达人源性抗角蛋白单链抗体。方法：从已构建好的质粒p3MH／ScFv中亚克隆目的片段ScFv并插入酵母表达载体p^PIC9K，构建成重组质粒p^PIC9K／ScFv，并测序鉴定。通过电转将重组质粒p^PIC9K／ScFv整合到巴氏毕赤酵母菌GS115的染色体上。经G418筛选得到高拷贝转化子及Mut表型鉴定后，用含0．5％甲醇的培养基诱导其分泌表达。结果：通过6天的诱导，该系统成功表达了抗角蛋白单链抗体，Western blot实验证实表达产物具有特异性。结论：获得了真核表达的抗角蛋白单链抗体，为其应用研究打下了基础。

Secretory expression of human ScFv against keratin in Pichia pastoris

Objective:To express secretively human anti-keratin ScFv in Pichia pastoris.Methods:Anti-keratin ScFv gene from plasmid p3MH/ScFv was subcloned into vector pPIC9K.After confirmed by DNA sequence analysis,the recombinant plasmid pPIC9K/ScFv was transducted into the genome of GS115 Pichia pastoris.Mut~s mutiple insert transformants were screened by G418 and induced by 5 ml/L methanol to express soluble ScFv.Results:After 6 days of methanol induction,anti-keratin ScFv was efficiently secreted into the medium.Western blot proved that the expressed protein had specific keratin binding activity.Conclusion:The successful expression of anti-keratin ScFv in Pichia pastoris laid a solid foundation for its further application.