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

作　　者：樊建勇[1] 王刚[2] 夏汝山[2] 陈宇萍[2] 高天文[2] 刘玉峰[2] 

机构地区：[1]广州军区广州总医院皮肤科,广州510010 [2]第四军医大学西京医院全军皮肤性病中心,西安710032

出　　处：《中国免疫学杂志》2005年第9期659-662,共4页Chinese Journal of Immunology

基　　金：国家863计划(2001AA215361);国家自然科学基金(30371650)资助项目

摘　　要：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.

关 键 词：单链抗体 角蛋白 毕赤酵母 分泌表达 

分 类 号：R392.11[医药卫生—免疫学]