人源性抗角蛋白单链抗体在巴氏毕赤酵母的分泌表达

Secretory expression of human ScFv against keratin in Pichia pastoris

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p3MHScFv中亚克隆目的片段ScFv并插入酵母表达载体pPIC9K,构建成重组质粒pPIC9KScFv,并测序鉴定。通过电转将重组质粒pPIC9KScFv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,Westernblot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。

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 pastoffs. 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 pastoffs laid a solid foundation for its further application.