人源性抗角蛋白抗体Fab段在巴氏毕赤酵母菌中的表达及表达条件的优化  

Expression of anti-keratin human Fab in Pichia pastoris and optimization of expression condition

作　　者：樊建勇[1] 王刚[1] 沈柱[1] 李承新[1] 高天文[1] 刘玉峰[1] 

机构地区：[1]第四军医大学西京医院全军皮肤性病中心

出　　处：《细胞与分子免疫学杂志》2006年第6期797-800,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家高技术研究发展计划(863)资助项目(2001AA215361);国家自然科学基金资助项目(30371650)

摘　　要：目的:用巴氏毕赤酵母表达抗角蛋白抗体Fab段并优化表达条件。方法:将抗角蛋白人Fab原核表达载体p3MH/Fab中的κ链和Fd段基因,分别亚克隆到酵母菌表达载体pPIC9K和pPICZαA中,构建成重组质粒pPIC9K/L和pPICZαA/Fd并测序鉴定。将重组质粒pPIC9K/L和pPICZαA/Fd分步整合到酵母菌GS115的染色体上,经G418和Zeocin筛选得到高拷贝的转化子。对Mut表型鉴定后,优化pH及甲醇浓度等表达条件。结果:优化表达条件后,成功地表达抗角蛋白Fab段抗体。Westernblot分析证实,表达产物具有良好的角蛋白结合活性和特异性。结论:人源性抗角蛋白Fab段抗体在巴氏毕赤酵母菌中获得成功表达,为其进一步的应用研究打下了基础。AIM： To express human anti-keratin Fab in Pichia pastoris secretively and optimize the expression condition. METHODS： Genes of κ chain and Fd fragment of anti-keratin antibody from the plasmids p3MH/Fab were subcloned into vectors pPIC9K and pPICZaA respectively. After confirmed by DNA sequence analysis, the recombinant plasmids pPIC9K/L and pPICZαA/Fd were transducted into the genome of GS115 Pichia pastoris using two-step integrating technology. Mut^＋ mutiple insert transformants were screened by G418 and Zeocin. The pH value and methanol concentration was adjusted to optimize the expression condition. RESULTS： Under the optimized expression condition, the Fab of anti-keratin antibody was efficiently secreted into the medium. Western blot assay proved that the expressed protein had specific keratin binding activity. CONCLUSION： The successful expression of the anti-keratin Fab in Pichia pastoris has laid a solid foundation for its further application.

关 键 词：FAB 角蛋白 毕赤酵母菌 分泌表达 

分 类 号：R392.11[医药卫生—免疫学]