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.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
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)
关键词
肝癌
单克隆抗体
cFab
毕赤酵母
分泌表达
hepatocellular carcinoma
monoclonal antibody
chimeric Fab
pichia parstoris
secretory expression
