抗人肝癌单克隆抗体嵌合Fab在巴氏毕赤酵母中的高效分泌表达  

Secretory expression of chimeric Fab antibody HAb18 against human hepatocellular carcinoma in Pichia pastoris

作　　者：董红霖[1] 邢金良[2] 安家泽 杨向民[2] 姚西英[2] 窦科峰[1] 陈志南[1] 

机构地区：[1]第四军医大学西京医院肝胆外科,陕西西安710032 [2]第四军医大学基础部细胞工程研究中心,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2005年第3期342-346,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家高技术研究发展计划(863)重点项目(No.2001AA2l5101)

摘　　要：目的:通过分步整合,用巴氏毕赤酵母表达抗人肝癌单克隆抗体(mAb)HAb18的嵌合Fab(cFab)片段。方法:将抗人肝癌mAbcFab/HAb18基因的原核表达载体pET32a/cFab中的CL和Fd段基因,分别亚克隆到酵母表达载体pPIC9K和pPICZαA中,构建重组质粒pPIC9K/CL和pPICZαA/Fd并测序鉴定。将重组质粒pPIC9K/CL和pPICZαA/Fd分步整合到酵母菌GS115的染色体上,经G418和Zeocin筛选高拷贝的转化子及鉴定Mut表型后,用含5mL/L甲醇的培养基诱导表达。结果:成功地表达抗人肝癌mAbHAb18的cFab,表达水平为26mg/L。Westernblot鉴定证实,表达产物具有良好的与HAb18结合的活性和特异性。结论:cFab/HAb18在巴氏毕赤酵母获得表达,为对其进一步大规模的生产和临床应用奠定了基础。AIM: To express secretively chimeric Fab antibody HAb18 (cFab)against human hepatocellular carcinoma in Pichia pastoris. METHODS: Genes encoding C_L chain and Fd fragment of cFab antibody HAb18 were subcloned into vectors pPIC9K and pPICZαA, respectively. After confirmed by DNA sequence analysis, the recombinant plasmids pPIC9K/C_L and pPICZαA/Fd were transformed into the genome of Pichia pastoris GS115. Mut^+ multiple insert transformants were screened by G418 and Zeocin and then induced with 5 mL/L methanol to express cFab. (RESULTS:) 4 days after methanol induction, 26 mg/L of the cFab fragment was detected in the culture supernatant. Western blot proved that the expressed protein could specifically bind with HAb18GEF antigen. CONCLUSION: The successful expression of cFab/HAb18 in Pichia pastoris lays the foundation for large-scale production and further application of the antibody.

关 键 词：肝癌 单克隆抗体 cFab 毕赤酵母 分泌表达 

分 类 号：R392.11[医药卫生—免疫学]